la pcr kit  (TaKaRa)

 
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    Name:
    LA PCR Kit
    Description:
    The LA PCR Kit Version 2 1 contains all the reagents needed for amplification of long DNA templates enabling routine amplification of products up to 20 kb in length including GC rich amplicons For some DNA templates amplification of up to 48 kb is possible This long range PCR kit contains TaKaRa LA Taq DNA Polymerase buffers MgCl2 dNTPs molecular weight markers and control templates plus corresponding primers to ensure optimal PCR performance during long PCR
    Catalog Number:
    rr013b
    Price:
    None
    Size:
    100 Rxns
    Category:
    LA Taq PCR kit LA Taq products Long range PCR PCR
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    Structured Review

    TaKaRa la pcr kit
    Identification of the mutant strain RA1062. A <t>PCR</t> amplification. M: <t>Takara</t> DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p
    The LA PCR Kit Version 2 1 contains all the reagents needed for amplification of long DNA templates enabling routine amplification of products up to 20 kb in length including GC rich amplicons For some DNA templates amplification of up to 48 kb is possible This long range PCR kit contains TaKaRa LA Taq DNA Polymerase buffers MgCl2 dNTPs molecular weight markers and control templates plus corresponding primers to ensure optimal PCR performance during long PCR
    https://www.bioz.com/result/la pcr kit/product/TaKaRa
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    la pcr kit - by Bioz Stars, 2020-09
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    1) Product Images from "The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis"

    Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

    Journal: Veterinary Research

    doi: 10.1186/s13567-018-0589-8

    Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p
    Figure Legend Snippet: Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Marker, Southern Blot, Positive Control, Negative Control, Agarose Gel Electrophoresis, Labeling, Real-time Polymerase Chain Reaction, Expressing

    2) Product Images from "The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori"

    Article Title: The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori

    Journal: Journal of Insect Science

    doi:

    Pre-mRNA transcribed from the Bmdsx mini gene is sex-specifically spliced. (A) The diagram shows the structure of the Bmdsx minigene. Open boxes: common exons. Shaded boxes: female-specific exons. Arrows: primers for PCR. (B) Poly (A)+RNA was extracted from the fat body of transgenic silkworms. These RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers TGM2F and TGM2R. Resulting products were separated on a 1% agarose gel and visualized with SYBR Green I (Molecular Probes) at a dilution of 1: 10,000. M represents the DNA marker(λ /Hind III+ φX174/ Hinc II). The bands for the PCR products are schematically shown. (C) Poly (A)+RNA was extracted from the fat body. Upper lanes: these RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers TGM2F and TGM2R. Lower lanes: these RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers BmA3QPCR1F and BmA3QPCR1R. Resulting products were separated on a 1% agarose gel and visualized with SYBR Green I (Molecular Probes) at a dilution of 1: 10,000 (upper lanes) and visualized with ethidium bromide (lower lanes). M represents the DNA marker (λ /Hind III+ φX174/ Hinc II). The bands for the PCR products are schematically shown.
    Figure Legend Snippet: Pre-mRNA transcribed from the Bmdsx mini gene is sex-specifically spliced. (A) The diagram shows the structure of the Bmdsx minigene. Open boxes: common exons. Shaded boxes: female-specific exons. Arrows: primers for PCR. (B) Poly (A)+RNA was extracted from the fat body of transgenic silkworms. These RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers TGM2F and TGM2R. Resulting products were separated on a 1% agarose gel and visualized with SYBR Green I (Molecular Probes) at a dilution of 1: 10,000. M represents the DNA marker(λ /Hind III+ φX174/ Hinc II). The bands for the PCR products are schematically shown. (C) Poly (A)+RNA was extracted from the fat body. Upper lanes: these RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers TGM2F and TGM2R. Lower lanes: these RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers BmA3QPCR1F and BmA3QPCR1R. Resulting products were separated on a 1% agarose gel and visualized with SYBR Green I (Molecular Probes) at a dilution of 1: 10,000 (upper lanes) and visualized with ethidium bromide (lower lanes). M represents the DNA marker (λ /Hind III+ φX174/ Hinc II). The bands for the PCR products are schematically shown.

    Techniques Used: Polymerase Chain Reaction, Transgenic Assay, Random Hexamer Labeling, Amplification, Agarose Gel Electrophoresis, SYBR Green Assay, Marker

    3) Product Images from "An Ebox Element in the Proximal Gata4 Promoter Is Required for Gata4 Expression In Vivo"

    Article Title: An Ebox Element in the Proximal Gata4 Promoter Is Required for Gata4 Expression In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029038

    Targeting of the Ebox in the mouse Gata4 promoter upstream of exon 1a. (A) Schematic representation of the Gata4 locus, targeting vector and final Cre-recombined Gata4 EboxKO allele. The targeting construct was designed to allow replacement of the Gata4 Ebox (white circle) by a Hind III restriction site. A neomycin cassette flanked by LoxP sites (black triangles) was also included to allow G418-mediated selection in ES cells. Black arrows indicate oligonucleotide primers and solid black bars indicate the location of the probes used for Southern blotting. H, Hind III; S, Sac I. (B) Southern blot analyses confirming the predicted targeting event on both the 5′ and 3′ side for an ES cell clone used to generate chimeras. Genomic DNA was analyzed using the indicated combination of restriction endonuclease and probe. MW, 1 kb DNA ladder; ES, ES cell genomic DNA. (C) PCR analysis of tail genomic DNA from F1 offspring using the primers 1 and 2 to amplify the wild-type allele and primers 1 and 3 to identify the mutated allele.
    Figure Legend Snippet: Targeting of the Ebox in the mouse Gata4 promoter upstream of exon 1a. (A) Schematic representation of the Gata4 locus, targeting vector and final Cre-recombined Gata4 EboxKO allele. The targeting construct was designed to allow replacement of the Gata4 Ebox (white circle) by a Hind III restriction site. A neomycin cassette flanked by LoxP sites (black triangles) was also included to allow G418-mediated selection in ES cells. Black arrows indicate oligonucleotide primers and solid black bars indicate the location of the probes used for Southern blotting. H, Hind III; S, Sac I. (B) Southern blot analyses confirming the predicted targeting event on both the 5′ and 3′ side for an ES cell clone used to generate chimeras. Genomic DNA was analyzed using the indicated combination of restriction endonuclease and probe. MW, 1 kb DNA ladder; ES, ES cell genomic DNA. (C) PCR analysis of tail genomic DNA from F1 offspring using the primers 1 and 2 to amplify the wild-type allele and primers 1 and 3 to identify the mutated allele.

    Techniques Used: Plasmid Preparation, Construct, Selection, Southern Blot, Polymerase Chain Reaction

    4) Product Images from "Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms"

    Article Title: Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007534

    siRNA (-2752-21) targets tomato SlLNR1 . (A) Schematic illustration of SlLNR1 structure and its associated siRNAs. The sense SlLNR1 , SlLNR1 (+), from the susceptible cultivar is an 1132-nt lncRNA, which is validated by RACE. Its anti-sense, SlLNR1 (-), is generated by sequencing a segmental RT-PCR (the 54 th bp of the 3’ end to the 1008 th ). The full sequence of SlLNR1 was shown in S4 Fig . The vertical bar indicate the target site of siRNA(-2752-21). The data derived from small RNA sequencing of TYLCV infected plants or mock was aligned to SlLNR1 and the number indicates the aligned siRNA reads. (B) Negative correlation of expression of siRNA(-2752-21) and SlLNR1 . SlLNR1 (+) or SlLNR1 (-) was expressed with siRNA(-2752-21) in N . benthamiana . The RNA sample was extracted at 48 h after agroinfiltration. The EF1a gene of N . benthamiana was used as a refernce. (C) SlLNR1 was down regulated in the pTRV2:IR inoculated susceptible plants but not in the resistant plants. The RNA sample was extracted at 15 days after pTRV2:IR and EV inoculated plants. The relative expression of SlLNR1 was measured by qRT-PCR and calculated in relation to EV inoculated plants according to the ΔΔ Ct method. The tomato actin gene was set as reference gene. Error bars represented SE of three biological replicates and significant differences by Student’s t test (*, p
    Figure Legend Snippet: siRNA (-2752-21) targets tomato SlLNR1 . (A) Schematic illustration of SlLNR1 structure and its associated siRNAs. The sense SlLNR1 , SlLNR1 (+), from the susceptible cultivar is an 1132-nt lncRNA, which is validated by RACE. Its anti-sense, SlLNR1 (-), is generated by sequencing a segmental RT-PCR (the 54 th bp of the 3’ end to the 1008 th ). The full sequence of SlLNR1 was shown in S4 Fig . The vertical bar indicate the target site of siRNA(-2752-21). The data derived from small RNA sequencing of TYLCV infected plants or mock was aligned to SlLNR1 and the number indicates the aligned siRNA reads. (B) Negative correlation of expression of siRNA(-2752-21) and SlLNR1 . SlLNR1 (+) or SlLNR1 (-) was expressed with siRNA(-2752-21) in N . benthamiana . The RNA sample was extracted at 48 h after agroinfiltration. The EF1a gene of N . benthamiana was used as a refernce. (C) SlLNR1 was down regulated in the pTRV2:IR inoculated susceptible plants but not in the resistant plants. The RNA sample was extracted at 15 days after pTRV2:IR and EV inoculated plants. The relative expression of SlLNR1 was measured by qRT-PCR and calculated in relation to EV inoculated plants according to the ΔΔ Ct method. The tomato actin gene was set as reference gene. Error bars represented SE of three biological replicates and significant differences by Student’s t test (*, p

    Techniques Used: Generated, Sequencing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, RNA Sequencing Assay, Infection, Expressing, Quantitative RT-PCR

    Identification of a 25-nt segment and a vsRNA that induce stunt and curled leaves in tomato. (A) VsRNAs generated by the IR and sequence alignment of vsRNAs and SlLNR1 . Location and frequency of TYCLV-derived siRNAs (vsRNAs) were mapped to the IR in sense- (above the x-axis) or antisense- (below the x-axis) orientation. Genome organization of the IR was shown at the top in which the inverted repeat was symbolized as a stem loop. Numbers indicate the first (2616) and last (147) nucleotides of the IR sequence. The histogram of location, frequency and size distribution of vsRNAs corresponding to the 25-nt-fragment (2730–2754) were shown at the medium panel. The fragment was highly complemented with SlLNR1 (-). The scissor means the cleavage site determined by 5’-RACE analysis. (B) Phenotypes of tomato inoculated with pTRV2:4TR. TYLCV-susceptible tomato plants were inoculated with pTRV2 containing 4×25-nt-fragment (2730–2754). The photos were taken at 15 dpi. (C) Validation of siRNA(-2752-21) in the tomato plants by siRNA Northern blot. The leaves of susceptible tomato plants inoculated by TYLCV infectious clone, natural infection by viruliferous whiteflies, agroinfiltrated with pTRV2:4TR and pTRV2:IR were used for total RNA extraction and analyzed at 24 dpi. U6 gene was set as the internal control. (D) Validation of siRNA(-2752-21) presence and downregulation of SlLNR1 in the overexpressed plants. Two individual transgenic lines (pCAMBIA2301:siRNA-1/2) with overexpression of siRNA(-2752-21) were used for total RNA extraction and analyzed. EV indicates the transgenic plant with the EV. The lower panel shows the relative expressi o n of SlLNR1 that was measured by qRT-PCR and calculated in relation to the transgenic plants according to the ΔΔ Ct method using tomat o actin gene as the reference. Error bars represented SE of three biological replicates and significant differences by Student’ s t test (*, p
    Figure Legend Snippet: Identification of a 25-nt segment and a vsRNA that induce stunt and curled leaves in tomato. (A) VsRNAs generated by the IR and sequence alignment of vsRNAs and SlLNR1 . Location and frequency of TYCLV-derived siRNAs (vsRNAs) were mapped to the IR in sense- (above the x-axis) or antisense- (below the x-axis) orientation. Genome organization of the IR was shown at the top in which the inverted repeat was symbolized as a stem loop. Numbers indicate the first (2616) and last (147) nucleotides of the IR sequence. The histogram of location, frequency and size distribution of vsRNAs corresponding to the 25-nt-fragment (2730–2754) were shown at the medium panel. The fragment was highly complemented with SlLNR1 (-). The scissor means the cleavage site determined by 5’-RACE analysis. (B) Phenotypes of tomato inoculated with pTRV2:4TR. TYLCV-susceptible tomato plants were inoculated with pTRV2 containing 4×25-nt-fragment (2730–2754). The photos were taken at 15 dpi. (C) Validation of siRNA(-2752-21) in the tomato plants by siRNA Northern blot. The leaves of susceptible tomato plants inoculated by TYLCV infectious clone, natural infection by viruliferous whiteflies, agroinfiltrated with pTRV2:4TR and pTRV2:IR were used for total RNA extraction and analyzed at 24 dpi. U6 gene was set as the internal control. (D) Validation of siRNA(-2752-21) presence and downregulation of SlLNR1 in the overexpressed plants. Two individual transgenic lines (pCAMBIA2301:siRNA-1/2) with overexpression of siRNA(-2752-21) were used for total RNA extraction and analyzed. EV indicates the transgenic plant with the EV. The lower panel shows the relative expressi o n of SlLNR1 that was measured by qRT-PCR and calculated in relation to the transgenic plants according to the ΔΔ Ct method using tomat o actin gene as the reference. Error bars represented SE of three biological replicates and significant differences by Student’ s t test (*, p

    Techniques Used: Generated, Sequencing, Derivative Assay, Northern Blot, Infection, RNA Extraction, Transgenic Assay, Over Expression, Quantitative RT-PCR

    5) Product Images from "Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy"

    Article Title: Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy

    Journal: Nature

    doi: 10.1038/nature10456

    An SVA retrotransposal insertion induces abnormal splicing in FCMD a, Expression analysis of various regions of fukutin mRNA in lymphoblasts. Gray bar, the ratio of RT-PCR product in FCMD patients relative to the normal control; Numbers on the X axis, nucleotide positions of both forward and reverse primers in fukutin . Error bars, s.e.m. b, Long range PCR using primers flanking the expression-decreasing area (nucleotide position 1061 to 5941) detected a 3-kb PCR product in FCMD lymphoblast cDNA (open arrow) and 8-kb product in FCMD genomic DNA (closed arrow). In the normal control, cDNA and genomic DNA both showed 5-kb PCR products. The 8-kb band was weak probably because VNTR region of SVA is GC-rich (82%). c, Schematic representation of genomic DNA and cDNA in FCMD. Black and white arrows, forward and reverse sequencing primers. The intronic sequence in FCMD is indicated in lower case. The authentic stop codon is colored in red, and the new stop codon is colored in blue. d, e, Northern blot analysis of fukutin in human lymphoblasts ( d ) and model mice ( e ). F, FCMD; N, nomal control. The wild-type mouse fukutin mRNA was detected at a size of 6.1 kb. Both skeletal muscle (left) and brain (right) showed smaller, abnormal bands (open arrows) in Hp/Hp mice. Wt, wild type; Hn, Hn/Hn mice; Hp, Hp/Hp mice. f, Schematic representation of genomic DNA and cDNA in ARH ( LDLRAP1 , left), NLSDM ( PNPLA2 , middle), and human ( AB627340 , right).
    Figure Legend Snippet: An SVA retrotransposal insertion induces abnormal splicing in FCMD a, Expression analysis of various regions of fukutin mRNA in lymphoblasts. Gray bar, the ratio of RT-PCR product in FCMD patients relative to the normal control; Numbers on the X axis, nucleotide positions of both forward and reverse primers in fukutin . Error bars, s.e.m. b, Long range PCR using primers flanking the expression-decreasing area (nucleotide position 1061 to 5941) detected a 3-kb PCR product in FCMD lymphoblast cDNA (open arrow) and 8-kb product in FCMD genomic DNA (closed arrow). In the normal control, cDNA and genomic DNA both showed 5-kb PCR products. The 8-kb band was weak probably because VNTR region of SVA is GC-rich (82%). c, Schematic representation of genomic DNA and cDNA in FCMD. Black and white arrows, forward and reverse sequencing primers. The intronic sequence in FCMD is indicated in lower case. The authentic stop codon is colored in red, and the new stop codon is colored in blue. d, e, Northern blot analysis of fukutin in human lymphoblasts ( d ) and model mice ( e ). F, FCMD; N, nomal control. The wild-type mouse fukutin mRNA was detected at a size of 6.1 kb. Both skeletal muscle (left) and brain (right) showed smaller, abnormal bands (open arrows) in Hp/Hp mice. Wt, wild type; Hn, Hn/Hn mice; Hp, Hp/Hp mice. f, Schematic representation of genomic DNA and cDNA in ARH ( LDLRAP1 , left), NLSDM ( PNPLA2 , middle), and human ( AB627340 , right).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Northern Blot, Mouse Assay

    AON cocktail rescues normal fukutin mRNA a, RT-PCR diagram of three primers designed to assess normal fukutin mRNA recovery (upper). Black closed arrow, a common forward primer located on fukutin coding region; black open arrow, a reverse primer to detect the abnormal RT-PCR product (161 bp); gray closed arrow, the other reverse primer to detect the restored normal RT-PCR product (129 bp). The effect on Hp/Hp ES cells treated with each single or a cocktail of AONs (lower). F, FCMD; N, normal sample. b, Rescue from abnormal splicing in VMO-treated in Hp/Hp mice and Hp/− mice. Local injection of AED cocktail into TA (n=3). Dys, a negative control. c, Rescue from abnormal splicing in VMO-treated human FCMD lymphoblasts (left, n=2) and myotubes (right, n=2). The Y axis shows the percent recovery of normal mRNA (* p
    Figure Legend Snippet: AON cocktail rescues normal fukutin mRNA a, RT-PCR diagram of three primers designed to assess normal fukutin mRNA recovery (upper). Black closed arrow, a common forward primer located on fukutin coding region; black open arrow, a reverse primer to detect the abnormal RT-PCR product (161 bp); gray closed arrow, the other reverse primer to detect the restored normal RT-PCR product (129 bp). The effect on Hp/Hp ES cells treated with each single or a cocktail of AONs (lower). F, FCMD; N, normal sample. b, Rescue from abnormal splicing in VMO-treated in Hp/Hp mice and Hp/− mice. Local injection of AED cocktail into TA (n=3). Dys, a negative control. c, Rescue from abnormal splicing in VMO-treated human FCMD lymphoblasts (left, n=2) and myotubes (right, n=2). The Y axis shows the percent recovery of normal mRNA (* p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Injection, Negative Control

    6) Product Images from "Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells"

    Article Title: Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.022590

    TAF1 and MTS transcript expression levels in fibroblasts. (A) Genomic DNA (gDNA) from all individuals was PCR amplified with primers flanking the insertion site to confirm the presence of the SVA. Lane 1: 1 kb DNA ladder. Lane 2: no template control (H 2 O). Lanes 3-7: XDP lines (left to right) 32517, 33109, 33363, 33808, 34363. Lanes 8-12: Control lines (left to right) 32643, 33113, 33114, 33809, 33362. The predicted 3229 bp SVA product was present in all XDP samples (upper arrow), whereas controls had a product of ∼599 bp (lower arrow), a difference consistent with the size of the SVA. (B) Quantitative expression analysis of TAF1 transcript fragments in XDP vs control fibroblasts ( n =5 each) based on comparative Ct method. Expression levels were normalized to the mean of housekeeping genes HPRT1 and TFRC . Levels of transcript fragments amplified by primer sets TA02-334, TAF1-3′, TA14-385N and TAF1-3′N were significantly lower in XDP vs control cells, whereas expression of the transcript amplified by TA09-693 was significantly increased in XDP vs control samples. The neural-specific transcript, N-TAF1, amplified by primer set TA14-391, as well as all six transcripts incorporating MTS sequences, were not detected in fibroblasts. Data represent mean fold changes±standard errors, analyzed by Student's t -test. * P
    Figure Legend Snippet: TAF1 and MTS transcript expression levels in fibroblasts. (A) Genomic DNA (gDNA) from all individuals was PCR amplified with primers flanking the insertion site to confirm the presence of the SVA. Lane 1: 1 kb DNA ladder. Lane 2: no template control (H 2 O). Lanes 3-7: XDP lines (left to right) 32517, 33109, 33363, 33808, 34363. Lanes 8-12: Control lines (left to right) 32643, 33113, 33114, 33809, 33362. The predicted 3229 bp SVA product was present in all XDP samples (upper arrow), whereas controls had a product of ∼599 bp (lower arrow), a difference consistent with the size of the SVA. (B) Quantitative expression analysis of TAF1 transcript fragments in XDP vs control fibroblasts ( n =5 each) based on comparative Ct method. Expression levels were normalized to the mean of housekeeping genes HPRT1 and TFRC . Levels of transcript fragments amplified by primer sets TA02-334, TAF1-3′, TA14-385N and TAF1-3′N were significantly lower in XDP vs control cells, whereas expression of the transcript amplified by TA09-693 was significantly increased in XDP vs control samples. The neural-specific transcript, N-TAF1, amplified by primer set TA14-391, as well as all six transcripts incorporating MTS sequences, were not detected in fibroblasts. Data represent mean fold changes±standard errors, analyzed by Student's t -test. * P

    Techniques Used: Expressing, Polymerase Chain Reaction, Amplification

    7) Product Images from "Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia"

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    Journal: Microbiology and Immunology

    doi: 10.1111/1348-0421.12648

    Estimated CTX prophage region structure of V. cholerae strain M25, the representative of group ET‐8. Profiles of CTX prophage region‐specific RFLP and PCR of strain M25 are unique and it was categorized as an independent group, ET‐8. The best estimated model for CTX prophage region of strain M25 is “TLC–RS1–CTX‐1–RS1–RTX” on chromosome I and there are no CTX prophage‐associated genes on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain M25, the representative of group ET‐8. Profiles of CTX prophage region‐specific RFLP and PCR of strain M25 are unique and it was categorized as an independent group, ET‐8. The best estimated model for CTX prophage region of strain M25 is “TLC–RS1–CTX‐1–RS1–RTX” on chromosome I and there are no CTX prophage‐associated genes on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C7, the representative of group ET‐6. Profiles of CTX prophage region‐specific RFLP and PCR of strain C7 are unique and it was categorized as an independent group, ET‐6. The best estimated model for CTX prophage region of strain C7 is “TLC– + CTX‐1– # CTX‐1– + CTX‐1– + CTX‐1–RTX” on chromosome I; no CTX prophage‐associated genes are present on chromosome II. + CTX‐1, CTX‐1 harboring SNPs in rstA (G301A), rstB (T84C), and in ctxA (G622A); # CTX‐1, CTX‐1 harboring SNPs in rstB (T84C) and in ctxA (G622A).
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain C7, the representative of group ET‐6. Profiles of CTX prophage region‐specific RFLP and PCR of strain C7 are unique and it was categorized as an independent group, ET‐6. The best estimated model for CTX prophage region of strain C7 is “TLC– + CTX‐1– # CTX‐1– + CTX‐1– + CTX‐1–RTX” on chromosome I; no CTX prophage‐associated genes are present on chromosome II. + CTX‐1, CTX‐1 harboring SNPs in rstA (G301A), rstB (T84C), and in ctxA (G622A); # CTX‐1, CTX‐1 harboring SNPs in rstB (T84C) and in ctxA (G622A).

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain P2, the representative of group ET‐5. Four V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐5. The strain P2 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain P2, the representative of group ET‐5. Four V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐5. The strain P2 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain P16, the representative of group ET‐7. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐7. The strain P16 was chosen as a representative and sequenced. The best estimated model for CTX prophage region of strain P16 is “TLC–CTX‐1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and “VCET1_GI–VCET1_GI” on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain P16, the representative of group ET‐7. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐7. The strain P16 was chosen as a representative and sequenced. The best estimated model for CTX prophage region of strain P16 is “TLC–CTX‐1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and “VCET1_GI–VCET1_GI” on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C1, the representative of group ET‐3. Profiles of CTX prophage region‐specific RFLP and PCR of strain C1 are unique and it was categorized as an independent group, ET‐3. The best estimated model for CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain C1, the representative of group ET‐3. Profiles of CTX prophage region‐specific RFLP and PCR of strain C1 are unique and it was categorized as an independent group, ET‐3. The best estimated model for CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain J6, the representative of group ET‐2. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR and were categorized into a group designated as ET‐2. The strain J6 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain J6, the representative of group ET‐2. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR and were categorized into a group designated as ET‐2. The strain J6 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C2, the representative of group ET‐4. Profiles of CTX prophage region‐specific RFLP and PCR of strain C2 are unique and it was categorized as an independent group, ET‐4. The best estimated model for CTX prophage region of strain C2 is “TLC–CTX‐1–RS1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and a single “VCET1_GI” on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain C2, the representative of group ET‐4. Profiles of CTX prophage region‐specific RFLP and PCR of strain C2 are unique and it was categorized as an independent group, ET‐4. The best estimated model for CTX prophage region of strain C2 is “TLC–CTX‐1–RS1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and a single “VCET1_GI” on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    8) Product Images from "Disruption of the Responsible Gene in a Phosphoglucomutase 1 Deficiency Patient by Homozygous Chromosomal Inversion"

    Article Title: Disruption of the Responsible Gene in a Phosphoglucomutase 1 Deficiency Patient by Homozygous Chromosomal Inversion

    Journal: JIMD Reports

    doi: 10.1007/8904_2018_108

    Disruption of the PGM1 gene in the study patient by a chromosomal inversion. ( a ) G-banding of the patient’s karyotype which was determined to be 46,XY,inv(1)(p31.1p32.3)x2, in which inv(1) was homozygous. ( b ) FISH signals for PGM1 (red arrow) are typically observed on the short arm of chromosome 1 in a normal karyotype. In contrast, the two distinctive signals were detected on the chromosome 1 arm in the study patient. ( c ) Schematic representation of the PGM1 gene structure. The blue boxes denote exons. The positions of the PCR primers are indicated by arrows. The position of the BAC probe is also indicated. ( d ) Agarose gel electrophoresis of long PCR products. 4F-4R and 4F3-4R primer pairs failed to amplify the PCR products in the study patient. P patient, C control, H H 2 O
    Figure Legend Snippet: Disruption of the PGM1 gene in the study patient by a chromosomal inversion. ( a ) G-banding of the patient’s karyotype which was determined to be 46,XY,inv(1)(p31.1p32.3)x2, in which inv(1) was homozygous. ( b ) FISH signals for PGM1 (red arrow) are typically observed on the short arm of chromosome 1 in a normal karyotype. In contrast, the two distinctive signals were detected on the chromosome 1 arm in the study patient. ( c ) Schematic representation of the PGM1 gene structure. The blue boxes denote exons. The positions of the PCR primers are indicated by arrows. The position of the BAC probe is also indicated. ( d ) Agarose gel electrophoresis of long PCR products. 4F-4R and 4F3-4R primer pairs failed to amplify the PCR products in the study patient. P patient, C control, H H 2 O

    Techniques Used: Fluorescence In Situ Hybridization, Polymerase Chain Reaction, BAC Assay, Agarose Gel Electrophoresis

    9) Product Images from "Amplification and next generation sequencing of near full-length human enteroviruses for identification and characterisation from clinical samples"

    Article Title: Amplification and next generation sequencing of near full-length human enteroviruses for identification and characterisation from clinical samples

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30322-y

    ( A ) Gel electrophoresis of near full-length genome PCR products produced from four long amplifying DNA polymerases; KlenTaq LA (discontinued), AccuTaq LA, PrimeSTAR GXL and Takara LA Taq (separate gel) per manufacturer’s instructions for samples with high GC/secondary structures. M, HyperLadder 1 kb; 1, CVB3 Nancy; 2, CVB5 Faulkner; 3, H 2 O control. ( B ) Gel electrophoresis of near full-length genome PCR products produced from Takara LA Taq DNA polymerase. M, HyperLadder 1 kb; 1–4, known EV positives from NSW Health Pathology East virology diagnostic lab; 5, CVB3 Nancy; 6, H 2 .
    Figure Legend Snippet: ( A ) Gel electrophoresis of near full-length genome PCR products produced from four long amplifying DNA polymerases; KlenTaq LA (discontinued), AccuTaq LA, PrimeSTAR GXL and Takara LA Taq (separate gel) per manufacturer’s instructions for samples with high GC/secondary structures. M, HyperLadder 1 kb; 1, CVB3 Nancy; 2, CVB5 Faulkner; 3, H 2 O control. ( B ) Gel electrophoresis of near full-length genome PCR products produced from Takara LA Taq DNA polymerase. M, HyperLadder 1 kb; 1–4, known EV positives from NSW Health Pathology East virology diagnostic lab; 5, CVB3 Nancy; 6, H 2 .

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Produced, Diagnostic Assay

    10) Product Images from "A Multiplex PCR Detection Assay for the Identification of Clinically Relevant Anaplasma Species in Field Blood Samples"

    Article Title: A Multiplex PCR Detection Assay for the Identification of Clinically Relevant Anaplasma Species in Field Blood Samples

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00606

    Optimization of the multiplex PCR components. (A) Annealing temperature gradients, M: DL2000 marker. Lanes 1–8: 64, 63, 62, 61, 60, 59, 58, and 57°C, respectively. (B) Dose of Anaplasma primers, lane M: DL2000 marker; Lanes 1–7: 0.08, 0.16, 0.24, 0.32, 0.4, 0.48, and 0.56 μM, respectively; Lane N: negative control. (C) La Taq DNA polymerase. M: DL2000 marker; Lanes 1–7: 0.75, 1.0, 1.25, 1.5, 1.75, 2, and 2.25 U, respectively; Lane N: negative control.
    Figure Legend Snippet: Optimization of the multiplex PCR components. (A) Annealing temperature gradients, M: DL2000 marker. Lanes 1–8: 64, 63, 62, 61, 60, 59, 58, and 57°C, respectively. (B) Dose of Anaplasma primers, lane M: DL2000 marker; Lanes 1–7: 0.08, 0.16, 0.24, 0.32, 0.4, 0.48, and 0.56 μM, respectively; Lane N: negative control. (C) La Taq DNA polymerase. M: DL2000 marker; Lanes 1–7: 0.75, 1.0, 1.25, 1.5, 1.75, 2, and 2.25 U, respectively; Lane N: negative control.

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Marker, Negative Control

    11) Product Images from "Segmental Maternal UPD of Chromosome 7q in a Patient With Pendred and Silver Russell Syndromes-Like Features"

    Article Title: Segmental Maternal UPD of Chromosome 7q in a Patient With Pendred and Silver Russell Syndromes-Like Features

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2018.00600

    Clinical and molecular characterization of the case. (A) Frontal and lateral view of the proband at the age of 36 months. Note the mild frontal bossing, low-set posteriorly rotated ears, and thin lips. Earing aids are in place. The patient also displays brachydactyly of both hands and feet and clinodactily of V finger of hands. Other clinical findings are listed in the side table, where 3 out of 6 NH-CSS for SRS are in bold characters. (B) The LR-PCR amplicons of the patient (P) and both her father (F) and mother (M) were resolved on a 0.8% agarose gel. The patient (P) displayed a unique shorter band of about 2.5 kb in size, the father (F) showed a unique band of over 10 kb in size, corresponding to the expected 14.7 kb wild type band, whereas her mother (M) showed two bands corresponding to the wild type and the deleted alleles. MW, molecular weight. (C) The proximal and distal breakpoints of the SLC26A4 intragenic deletion were mapped within SLC26A4 IVS16 and the IVS20, respectively. The deletion is about 13 kb long. Sequence alignments of the junction fragments revealed an insertion of a part of CCDC126 IVS3. The rejoining between SLC26A4 IVS20 and CCDC126 IVS3 distal bkp occurred through a de novo 3 bp GCC insertion. (D) Sequencing of the RT-PCR amplicons, extending from exons 13–14 to 3′UTR of SLC26A4 , confirmed the homozygous deletion of exons 17–20 in the child (P). The father (F) showed only the long transcript corresponding to the wild type pendrin, whereas the mother (M) displayed a short transcript and a long one, consistent with a heterozygous state of the deletion. MW, molecular weight.
    Figure Legend Snippet: Clinical and molecular characterization of the case. (A) Frontal and lateral view of the proband at the age of 36 months. Note the mild frontal bossing, low-set posteriorly rotated ears, and thin lips. Earing aids are in place. The patient also displays brachydactyly of both hands and feet and clinodactily of V finger of hands. Other clinical findings are listed in the side table, where 3 out of 6 NH-CSS for SRS are in bold characters. (B) The LR-PCR amplicons of the patient (P) and both her father (F) and mother (M) were resolved on a 0.8% agarose gel. The patient (P) displayed a unique shorter band of about 2.5 kb in size, the father (F) showed a unique band of over 10 kb in size, corresponding to the expected 14.7 kb wild type band, whereas her mother (M) showed two bands corresponding to the wild type and the deleted alleles. MW, molecular weight. (C) The proximal and distal breakpoints of the SLC26A4 intragenic deletion were mapped within SLC26A4 IVS16 and the IVS20, respectively. The deletion is about 13 kb long. Sequence alignments of the junction fragments revealed an insertion of a part of CCDC126 IVS3. The rejoining between SLC26A4 IVS20 and CCDC126 IVS3 distal bkp occurred through a de novo 3 bp GCC insertion. (D) Sequencing of the RT-PCR amplicons, extending from exons 13–14 to 3′UTR of SLC26A4 , confirmed the homozygous deletion of exons 17–20 in the child (P). The father (F) showed only the long transcript corresponding to the wild type pendrin, whereas the mother (M) displayed a short transcript and a long one, consistent with a heterozygous state of the deletion. MW, molecular weight.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Sequencing, Reverse Transcription Polymerase Chain Reaction

    12) Product Images from "Effects of rosuvastatin on atrial nerve sprouting and electrical remodeling in rabbits with myocardial infarction"

    Article Title: Effects of rosuvastatin on atrial nerve sprouting and electrical remodeling in rabbits with myocardial infarction

    Journal: International Journal of Clinical and Experimental Medicine

    doi:

    Effects of rosuvastatin on electrical remodeling after acute MI. The mRNA expression level of KCND3 potassium ion channel was detected by real-time PCR in atrial tissues following MI, in the sham, MI model, and intervention groups. Compared with the sham
    Figure Legend Snippet: Effects of rosuvastatin on electrical remodeling after acute MI. The mRNA expression level of KCND3 potassium ion channel was detected by real-time PCR in atrial tissues following MI, in the sham, MI model, and intervention groups. Compared with the sham

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Effects of rosuvastatin on TH expression level in atrial tissues after acute MI. The mRNA and protein expression levels of TH in rabbit atrial tissues were detected by real-Time PCR (A) and Western blot analysis (B), respectively. Compared with the sham
    Figure Legend Snippet: Effects of rosuvastatin on TH expression level in atrial tissues after acute MI. The mRNA and protein expression levels of TH in rabbit atrial tissues were detected by real-Time PCR (A) and Western blot analysis (B), respectively. Compared with the sham

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    13) Product Images from "Amplification and next generation sequencing of near full-length human enteroviruses for identification and characterisation from clinical samples"

    Article Title: Amplification and next generation sequencing of near full-length human enteroviruses for identification and characterisation from clinical samples

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30322-y

    ( A ) Gel electrophoresis of near full-length genome PCR products produced from four long amplifying DNA polymerases; KlenTaq LA (discontinued), AccuTaq LA, PrimeSTAR GXL and Takara LA Taq (separate gel) per manufacturer’s instructions for samples with high GC/secondary structures. M, HyperLadder 1 kb; 1, CVB3 Nancy; 2, CVB5 Faulkner; 3, H 2 O control. ( B ) Gel electrophoresis of near full-length genome PCR products produced from Takara LA Taq DNA polymerase. M, HyperLadder 1 kb; 1–4, known EV positives from NSW Health Pathology East virology diagnostic lab; 5, CVB3 Nancy; 6, H 2 O control. Full-length gels are presented in Supplementary Fig. 1 .
    Figure Legend Snippet: ( A ) Gel electrophoresis of near full-length genome PCR products produced from four long amplifying DNA polymerases; KlenTaq LA (discontinued), AccuTaq LA, PrimeSTAR GXL and Takara LA Taq (separate gel) per manufacturer’s instructions for samples with high GC/secondary structures. M, HyperLadder 1 kb; 1, CVB3 Nancy; 2, CVB5 Faulkner; 3, H 2 O control. ( B ) Gel electrophoresis of near full-length genome PCR products produced from Takara LA Taq DNA polymerase. M, HyperLadder 1 kb; 1–4, known EV positives from NSW Health Pathology East virology diagnostic lab; 5, CVB3 Nancy; 6, H 2 O control. Full-length gels are presented in Supplementary Fig. 1 .

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Produced, Diagnostic Assay

    14) Product Images from "Novel Endothelial Cell-Specific AQP1 Knockout Mice Confirm the Crucial Role of Endothelial AQP1 in Ultrafiltration during Peritoneal Dialysis"

    Article Title: Novel Endothelial Cell-Specific AQP1 Knockout Mice Confirm the Crucial Role of Endothelial AQP1 in Ultrafiltration during Peritoneal Dialysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145513

    Generation and validation of the floxed AQP1 allele. (A) Both 5' and 3' homologous recombinants were screened by long-range PCR with neo-specific primers P1F/1R and P2F/2R. PCR of representative embryonic stem (ES) clones showed the targeted 5'-(9,1kb) and the 3'- (5.3kb) homologous recombination events, respectively. (B) Targeted ES clones in (A) were confirmed by Southern blot analysis. Using the 5' probe (upper panel), the 3' probe (middle panel), and the neo probe (lower panel), the representative resulting Southern hybridisation signals appeared upon digestion of genomic DNA from ES clones with the Xho I (3' probe and neo probe) or Nsi I (5' probe). The genotypes of WT (+) and targeted ES clones with neo cassette (fl-neo) and the size of the detected fragments are indicated. Detection of a single 7.3 kb fragment with the neo probe indicates a singular integration event, whereas one clone (#) showed an additional integration of the neo cassette. Germline transmission was obtained from the clone indicated with an asterisk following blastocyst injection. (C) Genotyping of AQP1 +/+ (+), AQP1 fl-neo/+ (fl-neo) and AQP1 flox/+ (fl) mice by PCR using primers 3F and 3R. (D) WT (+/+), heterozygous (fl/+), homozygous (fl/fl) floxed alleles were distinguished by PCR with primers 4F and 4R. (E) Immunoblot of total protein fractions from visceral peritoneal (VP) homogenate probed with AQP1 antibody. Equal loading (40 μg of protein from each sample was verified using an anti-β-actin antibody. (F) AQP1 immunostaining showed normal localisation in the microvascular endothelium (arrows, stained in red) in VP and no apparent difference was observed between the AQP1 +/+ and AQP1 fl/fl mice. Calibration bar: 50μM.
    Figure Legend Snippet: Generation and validation of the floxed AQP1 allele. (A) Both 5' and 3' homologous recombinants were screened by long-range PCR with neo-specific primers P1F/1R and P2F/2R. PCR of representative embryonic stem (ES) clones showed the targeted 5'-(9,1kb) and the 3'- (5.3kb) homologous recombination events, respectively. (B) Targeted ES clones in (A) were confirmed by Southern blot analysis. Using the 5' probe (upper panel), the 3' probe (middle panel), and the neo probe (lower panel), the representative resulting Southern hybridisation signals appeared upon digestion of genomic DNA from ES clones with the Xho I (3' probe and neo probe) or Nsi I (5' probe). The genotypes of WT (+) and targeted ES clones with neo cassette (fl-neo) and the size of the detected fragments are indicated. Detection of a single 7.3 kb fragment with the neo probe indicates a singular integration event, whereas one clone (#) showed an additional integration of the neo cassette. Germline transmission was obtained from the clone indicated with an asterisk following blastocyst injection. (C) Genotyping of AQP1 +/+ (+), AQP1 fl-neo/+ (fl-neo) and AQP1 flox/+ (fl) mice by PCR using primers 3F and 3R. (D) WT (+/+), heterozygous (fl/+), homozygous (fl/fl) floxed alleles were distinguished by PCR with primers 4F and 4R. (E) Immunoblot of total protein fractions from visceral peritoneal (VP) homogenate probed with AQP1 antibody. Equal loading (40 μg of protein from each sample was verified using an anti-β-actin antibody. (F) AQP1 immunostaining showed normal localisation in the microvascular endothelium (arrows, stained in red) in VP and no apparent difference was observed between the AQP1 +/+ and AQP1 fl/fl mice. Calibration bar: 50μM.

    Techniques Used: Polymerase Chain Reaction, Homologous Recombination, Clone Assay, Southern Blot, Hybridization, Transmission Assay, Injection, Mouse Assay, Immunostaining, Staining

    Targeting construct and screening strategies. A part of the wild type (+) allele of mouse AQP1 is shown with indicated exons (black boxes) and restriction enzyme sites. The targeting allele (fl-neo) is indicated with 3’ and 5’ targeting arms (thick lines), loxP / FRT sites and pro- and eukaryotic neomycin selection cassette (neo-gb2-PGK). The 5’ probe and 3’ probe for Southern blot located outside the targeting vector detect 11.1-kb (+) and or 7.1-kb (fl-neo) fragments from Nsi I-digested genomic DNA and 22.6-kb (+) and or 7.3-kb (fl-neo) fragments following Xho I-digestion genomic DNA, respectively. Mice carrying the floxed allele (AQP fl-neo ) were crossed to FLPeR mice for excision of the FRT-flanked neo cassette. The resulting floxed mice (AQP fl ) were crossed to Cdh5 (PAC)-CreERT2 (Cdh5-Cre) transgenic mice to excise exons 2 and 3 following tamoxifen induction, and then generate the AQP1 null allele (AQP1 del ) in endothelial cells. The P1F/1R, P2F/2R, P3F/3R and P4F/4R primers for PCR-based genotype analyses and the lengths of their responding PCR products are indicated.
    Figure Legend Snippet: Targeting construct and screening strategies. A part of the wild type (+) allele of mouse AQP1 is shown with indicated exons (black boxes) and restriction enzyme sites. The targeting allele (fl-neo) is indicated with 3’ and 5’ targeting arms (thick lines), loxP / FRT sites and pro- and eukaryotic neomycin selection cassette (neo-gb2-PGK). The 5’ probe and 3’ probe for Southern blot located outside the targeting vector detect 11.1-kb (+) and or 7.1-kb (fl-neo) fragments from Nsi I-digested genomic DNA and 22.6-kb (+) and or 7.3-kb (fl-neo) fragments following Xho I-digestion genomic DNA, respectively. Mice carrying the floxed allele (AQP fl-neo ) were crossed to FLPeR mice for excision of the FRT-flanked neo cassette. The resulting floxed mice (AQP fl ) were crossed to Cdh5 (PAC)-CreERT2 (Cdh5-Cre) transgenic mice to excise exons 2 and 3 following tamoxifen induction, and then generate the AQP1 null allele (AQP1 del ) in endothelial cells. The P1F/1R, P2F/2R, P3F/3R and P4F/4R primers for PCR-based genotype analyses and the lengths of their responding PCR products are indicated.

    Techniques Used: Construct, Selection, Southern Blot, Plasmid Preparation, Mouse Assay, Transgenic Assay, Polymerase Chain Reaction

    15) Product Images from "A novel active endogenous retrovirus family contributes to genome variability in rat inbred strains"

    Article Title: A novel active endogenous retrovirus family contributes to genome variability in rat inbred strains

    Journal: Genome Research

    doi: 10.1101/gr.100073.109

    Insertion of a retrovirus into Cntrob . ( A ) Long-range PCR spanning exons 10 and 11 shows a ∼10-kb insertion in hd/hd homozygotes (lane 1 ), whereas the SHR control PCR product (lane 2 ) has the expected size (1578 bp). The Cntrob gene contains insertion
    Figure Legend Snippet: Insertion of a retrovirus into Cntrob . ( A ) Long-range PCR spanning exons 10 and 11 shows a ∼10-kb insertion in hd/hd homozygotes (lane 1 ), whereas the SHR control PCR product (lane 2 ) has the expected size (1578 bp). The Cntrob gene contains insertion

    Techniques Used: Polymerase Chain Reaction

    Phylogenetic characterization of the RnERV-K8e family. ( A ) Phylogeny of retroviruses based on the neighbor-joining method using highly conserved nucleotide sequence regions of the pol ) rooted using a non-LTR retrotransposon.
    Figure Legend Snippet: Phylogenetic characterization of the RnERV-K8e family. ( A ) Phylogeny of retroviruses based on the neighbor-joining method using highly conserved nucleotide sequence regions of the pol ) rooted using a non-LTR retrotransposon.

    Techniques Used: Sequencing

    Model of RnERV-K8e life cycle. An infectious retrovirus integrates into the germline of a host organism and is transmitted vertically. Such ERV may retain the capability to produce infectious particles with occasional re-infection or horizontal transmission.
    Figure Legend Snippet: Model of RnERV-K8e life cycle. An infectious retrovirus integrates into the germline of a host organism and is transmitted vertically. Such ERV may retain the capability to produce infectious particles with occasional re-infection or horizontal transmission.

    Techniques Used: Infection, Transmission Assay

    16) Product Images from "Fancd2 in vivo interaction network reveals a non-canonical role in mitochondrial function"

    Article Title: Fancd2 in vivo interaction network reveals a non-canonical role in mitochondrial function

    Journal: Scientific Reports

    doi: 10.1038/srep45626

    Dysregulation of mitochondrial genes in the absence of Fancd2 or Fanca. ( A ) Transmission electron microscopy of spleen MNCs and MEFs from WT, Fanca −/− and Fancd2 −/− mice. Swollen mitochondria with disorganized cristae are indicated with a white arrow. ( B ) The mtDNA copy number in bone marrow MNCs and spleen MNCs was analyzed by QPCR using primers against mtDNA and nuclear DNA (3 mice for each genotype). No significant difference was observed between the one-year old WT, Fanca −/− and Fancd2 −/− mice with Student’s t-test analysis. ( C ) To analyze mtDNA integrity in FA mice, 16 kb mtDNA of 2 month old mice spleen MNCs was amplified. The PCR product was subjected to small genome sequencing. The Fancd2 −/− mtDNA shows slightly higher mutation rates compared to WT, Fancd2 KI / KI and Fanca −/− mice, especially in the 16 s/12 s rRNA region. ( D ) Quantitative PCR analysis shows up-regulation of genes associated with mitochondrial biogenesis (Atad3, Tufm, Tfam) and down-regulation of genes involved in mitochondrial oxidative phosphorylation (Atp1a1, Atp2a2, Scl25a5) in Fanca −/− and Fancd2 −/− spleen MNCs compared to WT spleen MNC cells. The data represent a summary of more than three mice of each genotype from two independent experiments. The P values indicated were analyzed using Student’s t-test analysis. ( E ) For mtDNA-encoded genes, quantitative PCR analysis shows up-regulation of the 16 s rRNA and 12 s rRNA and down-regulation of Cox-1 and Nd2 expression in the Fanca −/− and Fancd2 −/− spleen MNCs comparing to WT cells. ( F ) Western blot shows increased expression of Atad3, Tufm, Aifm in the mitochondrion fraction from Fanca −/− and Fancd2 −/− spleen MNCs. ( G ) Western blot shows decreased Cox-1 expression in the mitochondrion fractions of Fanca −/− and Fancd2 −/− spleen MNCs. Full-length blot is presented in Supplementary Fig. 5 .
    Figure Legend Snippet: Dysregulation of mitochondrial genes in the absence of Fancd2 or Fanca. ( A ) Transmission electron microscopy of spleen MNCs and MEFs from WT, Fanca −/− and Fancd2 −/− mice. Swollen mitochondria with disorganized cristae are indicated with a white arrow. ( B ) The mtDNA copy number in bone marrow MNCs and spleen MNCs was analyzed by QPCR using primers against mtDNA and nuclear DNA (3 mice for each genotype). No significant difference was observed between the one-year old WT, Fanca −/− and Fancd2 −/− mice with Student’s t-test analysis. ( C ) To analyze mtDNA integrity in FA mice, 16 kb mtDNA of 2 month old mice spleen MNCs was amplified. The PCR product was subjected to small genome sequencing. The Fancd2 −/− mtDNA shows slightly higher mutation rates compared to WT, Fancd2 KI / KI and Fanca −/− mice, especially in the 16 s/12 s rRNA region. ( D ) Quantitative PCR analysis shows up-regulation of genes associated with mitochondrial biogenesis (Atad3, Tufm, Tfam) and down-regulation of genes involved in mitochondrial oxidative phosphorylation (Atp1a1, Atp2a2, Scl25a5) in Fanca −/− and Fancd2 −/− spleen MNCs compared to WT spleen MNC cells. The data represent a summary of more than three mice of each genotype from two independent experiments. The P values indicated were analyzed using Student’s t-test analysis. ( E ) For mtDNA-encoded genes, quantitative PCR analysis shows up-regulation of the 16 s rRNA and 12 s rRNA and down-regulation of Cox-1 and Nd2 expression in the Fanca −/− and Fancd2 −/− spleen MNCs comparing to WT cells. ( F ) Western blot shows increased expression of Atad3, Tufm, Aifm in the mitochondrion fraction from Fanca −/− and Fancd2 −/− spleen MNCs. ( G ) Western blot shows decreased Cox-1 expression in the mitochondrion fractions of Fanca −/− and Fancd2 −/− spleen MNCs. Full-length blot is presented in Supplementary Fig. 5 .

    Techniques Used: Transmission Assay, Electron Microscopy, Mouse Assay, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Sequencing, Mutagenesis, Expressing, Western Blot

    17) Product Images from "Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease"

    Article Title: Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2018020180

    MUC1 VNTR sequencing identifies novel mutations causing ADTKD- MUC1 . (A) Sequence logo showing the most conserved regions of the VNTR repeats. Corresponding amino acid sequences of wild-type MUC1 (wt_AA) and MUC1 fs (mut_AA) are shown below. To find novel frameshift mutations that change the open reading frame, different conserved 10-mers of the wild-type repeat were used as sequence anchors (underlined DNA sequence as an example). For each anchor pair, all sequences delimited by these two anchors that are changing an open reading frame ( i.e. , adding or deleting nucleotides) were selected from the FASTQ file. (B) Sequences of the canonical 60 nucleotide long wild-type VNTR repeat (wt) and candidate frameshift mutations identified in this study. (C) Random mutations are generated in DNA molecules during PCR amplification step. To find true germline mutations, the percentage of reads with a given sequence (putative frameshift mutation) from all reads was calculated for each of the analyzed samples ( y -axis), and this needed to be higher than the average+2 SD of the nine wild-type control samples. Indicated are numbers of controls (wt), patients with individual MUC1 mutations (27dupC, 28dupA, 26_27insG, 1–16dup, 23delinsAT, 51dupC), and individuals with still unknown MUC1 mutation(s) who have urinary cell smears positive for MUC1fs and who tested negative for 27dupC by conventional genotyping assay (unknown). (D) 27dupC, confirmed by a mass spectrometry-based primer extension assay. The 27dupC extension product is observed at 5904 D (red asterisk). (E) 28dupA, confirmed by a mass spectrometry-based assay. The 28dupA extension product is observed at 6571 D (red asterisk). (F) 26_27insG, confirmed by a mass spectrometry-based assay. The 26_27insG extension product is observed at 5944.85 D (red arrow). (G) 1–16dup confirmed by restriction analysis. The mutation creates new restriction site for Eci I enzyme. The electrophoretogram shows amplified VNTR regions of the affected patient (P1), two healthy relatives (H1, H2), and one unrelated control (NC) after (EciI) and before restriction by Eci I (PCR). The patient’s (P1) mutated allele (5000 bp) was cut into two fragments of 3000 and 2000 bp. (H) 23delinsAT, confirmed by restriction analysis. The mutation creates new restriction site for Fok I enzyme. The electrophoretogram is showing amplified VNTR regions of two affected patients (P1, P2) and one unrelated control (NC) after (FokI) and before restriction by Fok I (PCR). The patients’ (P1, P2) mutated alleles (3000 bp) were cut into two fragments of 2000 and 1000 bp.
    Figure Legend Snippet: MUC1 VNTR sequencing identifies novel mutations causing ADTKD- MUC1 . (A) Sequence logo showing the most conserved regions of the VNTR repeats. Corresponding amino acid sequences of wild-type MUC1 (wt_AA) and MUC1 fs (mut_AA) are shown below. To find novel frameshift mutations that change the open reading frame, different conserved 10-mers of the wild-type repeat were used as sequence anchors (underlined DNA sequence as an example). For each anchor pair, all sequences delimited by these two anchors that are changing an open reading frame ( i.e. , adding or deleting nucleotides) were selected from the FASTQ file. (B) Sequences of the canonical 60 nucleotide long wild-type VNTR repeat (wt) and candidate frameshift mutations identified in this study. (C) Random mutations are generated in DNA molecules during PCR amplification step. To find true germline mutations, the percentage of reads with a given sequence (putative frameshift mutation) from all reads was calculated for each of the analyzed samples ( y -axis), and this needed to be higher than the average+2 SD of the nine wild-type control samples. Indicated are numbers of controls (wt), patients with individual MUC1 mutations (27dupC, 28dupA, 26_27insG, 1–16dup, 23delinsAT, 51dupC), and individuals with still unknown MUC1 mutation(s) who have urinary cell smears positive for MUC1fs and who tested negative for 27dupC by conventional genotyping assay (unknown). (D) 27dupC, confirmed by a mass spectrometry-based primer extension assay. The 27dupC extension product is observed at 5904 D (red asterisk). (E) 28dupA, confirmed by a mass spectrometry-based assay. The 28dupA extension product is observed at 6571 D (red asterisk). (F) 26_27insG, confirmed by a mass spectrometry-based assay. The 26_27insG extension product is observed at 5944.85 D (red arrow). (G) 1–16dup confirmed by restriction analysis. The mutation creates new restriction site for Eci I enzyme. The electrophoretogram shows amplified VNTR regions of the affected patient (P1), two healthy relatives (H1, H2), and one unrelated control (NC) after (EciI) and before restriction by Eci I (PCR). The patient’s (P1) mutated allele (5000 bp) was cut into two fragments of 3000 and 2000 bp. (H) 23delinsAT, confirmed by restriction analysis. The mutation creates new restriction site for Fok I enzyme. The electrophoretogram is showing amplified VNTR regions of two affected patients (P1, P2) and one unrelated control (NC) after (FokI) and before restriction by Fok I (PCR). The patients’ (P1, P2) mutated alleles (3000 bp) were cut into two fragments of 2000 and 1000 bp.

    Techniques Used: Sequencing, Generated, Polymerase Chain Reaction, Amplification, Mutagenesis, Genotyping Assay, Mass Spectrometry, Primer Extension Assay

    18) Product Images from "A novel human in vitro papillomavirus type 16 positive tonsil cancer cell line with high sensitivity to radiation and cisplatin"

    Article Title: A novel human in vitro papillomavirus type 16 positive tonsil cancer cell line with high sensitivity to radiation and cisplatin

    Journal: BMC Cancer

    doi: 10.1186/s12885-019-5469-8

    a . Schematic representation of the HPV16 genome. Rectangles represent open reading frames, promoters p97 and p670 are indicated as arrows, and filled and open triangles represent 5′- and 3′-splices sites respectively [ 39 ]. HPV16 early and late polyA signals pAE and pAL are indicated. The mRNAs produced by HPV16 cells are indicated below the genome and RT-PCR primers are indicated as arrows and numbered. RT-PCR primers are listed in Table 1 . b . Total RNA extracted from LU-HNSCC-26 cells. 28S and 18S ribosomal RNAs are indicated. C. RT-PCR on total RNA extracted from the LU-HNSCC-26 cell line with the indicated primer pairs. D. Western blot with antibody against the HPV16 L1 or L2 protein on cell extracts from 293 T cells, 293 T cells transfected with a codon modified HPV16 L1 expression plasmid or LU-HNSCC-26 cells
    Figure Legend Snippet: a . Schematic representation of the HPV16 genome. Rectangles represent open reading frames, promoters p97 and p670 are indicated as arrows, and filled and open triangles represent 5′- and 3′-splices sites respectively [ 39 ]. HPV16 early and late polyA signals pAE and pAL are indicated. The mRNAs produced by HPV16 cells are indicated below the genome and RT-PCR primers are indicated as arrows and numbered. RT-PCR primers are listed in Table 1 . b . Total RNA extracted from LU-HNSCC-26 cells. 28S and 18S ribosomal RNAs are indicated. C. RT-PCR on total RNA extracted from the LU-HNSCC-26 cell line with the indicated primer pairs. D. Western blot with antibody against the HPV16 L1 or L2 protein on cell extracts from 293 T cells, 293 T cells transfected with a codon modified HPV16 L1 expression plasmid or LU-HNSCC-26 cells

    Techniques Used: Produced, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Modification, Expressing, Plasmid Preparation

    Coverage plot of the de novo HPV16 sequence of LU-HNSCC-26. Coverage was at minimum 134
    Figure Legend Snippet: Coverage plot of the de novo HPV16 sequence of LU-HNSCC-26. Coverage was at minimum 134

    Techniques Used: Sequencing

    Amplicons of long range HPV16 PCR from established cell line from a tonsil carcinoma at passage 2 (lane 1) and 8 (lanes 2–5 show 2-fold dilution series of template input to PCR). Lane 1: passage 2, input 15 ng, lane 2: passage 8, 37 ng, lane 3: passage 8, 18 ng, lane 4: passage 8, 9 ng. lane 5: passage 8, 4.5 ng. Lane 6: positive control, 5000 copies of cloned HPV16. Lane 7: negative control, water. Wm: Gene Ruler 100–10,000 bp (Thermo Fisher Scientific). The arrow indicates the position of an amplicon of 7900 bp
    Figure Legend Snippet: Amplicons of long range HPV16 PCR from established cell line from a tonsil carcinoma at passage 2 (lane 1) and 8 (lanes 2–5 show 2-fold dilution series of template input to PCR). Lane 1: passage 2, input 15 ng, lane 2: passage 8, 37 ng, lane 3: passage 8, 18 ng, lane 4: passage 8, 9 ng. lane 5: passage 8, 4.5 ng. Lane 6: positive control, 5000 copies of cloned HPV16. Lane 7: negative control, water. Wm: Gene Ruler 100–10,000 bp (Thermo Fisher Scientific). The arrow indicates the position of an amplicon of 7900 bp

    Techniques Used: Polymerase Chain Reaction, Positive Control, Clone Assay, Negative Control, Amplification

    19) Product Images from "The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity"

    Article Title: The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082149

    Structures of the pre- and post-Cre floxed mouse Zip5 gene and integration and genotyping screen designs. ( A ) The mouse Zip5 gene was captured using gap-repair and manipulated using galK recombineering. Exons ( 1–12 ) and the exon encoding transmembrane domain 1 ( TMD1 ) are indicated, as are the positions of LoxP sites (intron 4 and downstream of PGK Neo ), the PGK-neomycin ( PGK Neo ) cassette and the locations of primers used for genotyping. The LoxP site in intron 4 is flanked by an EcoRV restriction enzyme cleavage site. ( B ) The structure of the Zip5 gene after Cre recombination is shown. Recombination eliminates the transmembrane domain of ZIP5. ( C ) The floxed Zip5 gene was targeted into E14 ES cells and properly targeted ES cells were identified by long range PCR using flanking and internal primers. PCR products from the wild-type ( Wt ) and floxed ( Fx ) alleles are indicated. EcoRV cleavage was used to differentiate between the floxed and wild-type alleles in the 5′ PCR screen whereas the 3′ PCR screen yielded the predicted larger product from the wild-type allele. Targeted ES cells were used to generate mice homozygous for the floxed Zip5 allele. ( D ) Mice were genotyped by PCR amplification of the intron 4 region containing the LoxP site. The PCR product from homozygous Zip5 floxed mice before Cre-induced recombination is shown in the left lane while that from control mice is shown in the center lane and that from Zip5 -knockout mice ( Ko ) is shown in the right lane . For the intestine- and pancreas-specific knockout mice, detection of Zip5 mRNA and/or protein was employed to monitor the efficacy of recombination.
    Figure Legend Snippet: Structures of the pre- and post-Cre floxed mouse Zip5 gene and integration and genotyping screen designs. ( A ) The mouse Zip5 gene was captured using gap-repair and manipulated using galK recombineering. Exons ( 1–12 ) and the exon encoding transmembrane domain 1 ( TMD1 ) are indicated, as are the positions of LoxP sites (intron 4 and downstream of PGK Neo ), the PGK-neomycin ( PGK Neo ) cassette and the locations of primers used for genotyping. The LoxP site in intron 4 is flanked by an EcoRV restriction enzyme cleavage site. ( B ) The structure of the Zip5 gene after Cre recombination is shown. Recombination eliminates the transmembrane domain of ZIP5. ( C ) The floxed Zip5 gene was targeted into E14 ES cells and properly targeted ES cells were identified by long range PCR using flanking and internal primers. PCR products from the wild-type ( Wt ) and floxed ( Fx ) alleles are indicated. EcoRV cleavage was used to differentiate between the floxed and wild-type alleles in the 5′ PCR screen whereas the 3′ PCR screen yielded the predicted larger product from the wild-type allele. Targeted ES cells were used to generate mice homozygous for the floxed Zip5 allele. ( D ) Mice were genotyped by PCR amplification of the intron 4 region containing the LoxP site. The PCR product from homozygous Zip5 floxed mice before Cre-induced recombination is shown in the left lane while that from control mice is shown in the center lane and that from Zip5 -knockout mice ( Ko ) is shown in the right lane . For the intestine- and pancreas-specific knockout mice, detection of Zip5 mRNA and/or protein was employed to monitor the efficacy of recombination.

    Techniques Used: Polymerase Chain Reaction, Mouse Assay, Amplification, Knock-Out

    20) Product Images from "Myozenin: An ?-actinin- and ?-filamin-binding protein of skeletal muscle Z lines"

    Article Title: Myozenin: An ?-actinin- and ?-filamin-binding protein of skeletal muscle Z lines

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Subcellular localization of myozenin in human skeletal muscle. ( A ) Confocal indirect immunofluorescence microscopy of human skeletal muscle by using antimyozenin antisera showing striated staining pattern characteristic of sarcomeric proteins. ( B and C ) Immunogold transmission electron microscopy for myozenin ( B ) and α-actinin-2 ( C ) in human skeletal muscle showing predominant labeling at Z lines. ( D and E ) Double-label indirect immunofluorescence microscopy for myozenin (red) and myosin ( D , green) or tropomyosin ( E , green) in cultured human myotubes. ( F ) Quantitative grain measurements showing distribution of α-actinin-2 centered in middle of Z line ( Right ) and myozenin slightly skewed toward edges of Z lines ( Left ).
    Figure Legend Snippet: Subcellular localization of myozenin in human skeletal muscle. ( A ) Confocal indirect immunofluorescence microscopy of human skeletal muscle by using antimyozenin antisera showing striated staining pattern characteristic of sarcomeric proteins. ( B and C ) Immunogold transmission electron microscopy for myozenin ( B ) and α-actinin-2 ( C ) in human skeletal muscle showing predominant labeling at Z lines. ( D and E ) Double-label indirect immunofluorescence microscopy for myozenin (red) and myosin ( D , green) or tropomyosin ( E , green) in cultured human myotubes. ( F ) Quantitative grain measurements showing distribution of α-actinin-2 centered in middle of Z line ( Right ) and myozenin slightly skewed toward edges of Z lines ( Left ).

    Techniques Used: Immunofluorescence, Microscopy, Staining, Transmission Assay, Electron Microscopy, Labeling, Cell Culture

    Blot-overlay analysis of myozenin binding to α-actinins. 35 S-labeled probes are indicated below and nonradioactive IVTT products ( A and B ) or whole-tissue homogenates ( C ) loaded in each lane are indicated above A – C . ( A ) Myozenin binds to both α-actinin-2 and -3 but not to itself ( Left ), whereas α-actinin-2 and -3 both bind to myozenin as well as to themselves ( Center and Right ). Binding to α-actinin in myozenin lanes likely represents binding to α-actinin-1 and -4 produced from contaminating ACTN1 and ACTN4 mRNA in reticulocyte lysates. ( B ) Control experiment showing lack of myozenin binding to various partial dystrophin fragments: N828, N-terminal nos. 1–828; H12–R11, hinge no. 2–repeat no. 11; R7–R15, repeat nos. 7–15; R12–H13, repeat no. 12–hinge no. 3; and R19–H14, repeat no. 19–hinge no. 4. Parallel radiolabeled IVTT products of all clones are shown below to confirm production of expected size products. ( C ) Detection of myozenin-binding partners in human tissues by blot-overlay assay. Predominant binding is to a ≈100-kDa protein at expected size for α-actinin.
    Figure Legend Snippet: Blot-overlay analysis of myozenin binding to α-actinins. 35 S-labeled probes are indicated below and nonradioactive IVTT products ( A and B ) or whole-tissue homogenates ( C ) loaded in each lane are indicated above A – C . ( A ) Myozenin binds to both α-actinin-2 and -3 but not to itself ( Left ), whereas α-actinin-2 and -3 both bind to myozenin as well as to themselves ( Center and Right ). Binding to α-actinin in myozenin lanes likely represents binding to α-actinin-1 and -4 produced from contaminating ACTN1 and ACTN4 mRNA in reticulocyte lysates. ( B ) Control experiment showing lack of myozenin binding to various partial dystrophin fragments: N828, N-terminal nos. 1–828; H12–R11, hinge no. 2–repeat no. 11; R7–R15, repeat nos. 7–15; R12–H13, repeat no. 12–hinge no. 3; and R19–H14, repeat no. 19–hinge no. 4. Parallel radiolabeled IVTT products of all clones are shown below to confirm production of expected size products. ( C ) Detection of myozenin-binding partners in human tissues by blot-overlay assay. Predominant binding is to a ≈100-kDa protein at expected size for α-actinin.

    Techniques Used: Binding Assay, Labeling, Produced, Clone Assay, Overlay Assay

    CoIP of IVTT myozenin, α-actinin, and γ-filamin. Presence (+) or absence (−) of 35 S-labeled IVTT products in each reaction is shown above and precipitating Abs are indicated below. Identity of each precipitated protein is indicated ( Middle ). A and B illustrate CoIP of full-length α-actinins-2 and -3, respectively, with FLAG-tagged full-length myozenin by using either anti-α-actinin or anti-FLAG Abs (lanes 2 and 3). Each Ab is specific (lanes 4 and 5); however, there is some degree of aggregation and nonspecific precipitation of IVTT myozenin (lanes 1 and 5). ( C ) CoIP of myozenin, α-actinin, and γ-filamin (FLNC 2191–2705 ) by using specific antimyozenin and antifilamin Abs. Myozenin coimmunoprecipitates both α-actinin-2 (lane 1) and γ-filamin (lane 3). When equal volumes of all three IVTT products are combined, α-actinin CoIP predominates (lane 4). Use of anti-γ-filamin-precipitating Abs results in CoIP of myozenin but not α-actinin (lanes 5–8). Similarly, use of anti-α-actinin Abs coimmunoprecipitated myozenin but not γ-filamin (not shown), indicating a lack of direct interaction between α-actinin and γ-filamin. ( D ) Competitive CoIP of α-actinin and γ-filamin (FLNC 2191–2705 ) by using specific antimyozenin Abs. Increasing relative amounts of α-actinin (lanes 1–6 contain 0-, 1-, 2-, 4-, 10-, and 20-fold levels, respectively) competitively decreased CoIP of γ-filamin. Similarly, increasing amounts of γ-filamin reduced CoIP of α-actinin (not shown).
    Figure Legend Snippet: CoIP of IVTT myozenin, α-actinin, and γ-filamin. Presence (+) or absence (−) of 35 S-labeled IVTT products in each reaction is shown above and precipitating Abs are indicated below. Identity of each precipitated protein is indicated ( Middle ). A and B illustrate CoIP of full-length α-actinins-2 and -3, respectively, with FLAG-tagged full-length myozenin by using either anti-α-actinin or anti-FLAG Abs (lanes 2 and 3). Each Ab is specific (lanes 4 and 5); however, there is some degree of aggregation and nonspecific precipitation of IVTT myozenin (lanes 1 and 5). ( C ) CoIP of myozenin, α-actinin, and γ-filamin (FLNC 2191–2705 ) by using specific antimyozenin and antifilamin Abs. Myozenin coimmunoprecipitates both α-actinin-2 (lane 1) and γ-filamin (lane 3). When equal volumes of all three IVTT products are combined, α-actinin CoIP predominates (lane 4). Use of anti-γ-filamin-precipitating Abs results in CoIP of myozenin but not α-actinin (lanes 5–8). Similarly, use of anti-α-actinin Abs coimmunoprecipitated myozenin but not γ-filamin (not shown), indicating a lack of direct interaction between α-actinin and γ-filamin. ( D ) Competitive CoIP of α-actinin and γ-filamin (FLNC 2191–2705 ) by using specific antimyozenin Abs. Increasing relative amounts of α-actinin (lanes 1–6 contain 0-, 1-, 2-, 4-, 10-, and 20-fold levels, respectively) competitively decreased CoIP of γ-filamin. Similarly, increasing amounts of γ-filamin reduced CoIP of α-actinin (not shown).

    Techniques Used: Co-Immunoprecipitation Assay, Labeling

    Indirect immunofluorescence analysis of α-actinin and myozenin in nemaline rods. Longitudinal ( A ) and cross sections ( B ) stained with antimyozenin Ab. ( C ) Cross section stained with anti-α-actinin-2 Ab. All Abs stain both Z lines as well as nemaline rods.
    Figure Legend Snippet: Indirect immunofluorescence analysis of α-actinin and myozenin in nemaline rods. Longitudinal ( A ) and cross sections ( B ) stained with antimyozenin Ab. ( C ) Cross section stained with anti-α-actinin-2 Ab. All Abs stain both Z lines as well as nemaline rods.

    Techniques Used: Immunofluorescence, Staining

    Schematic diagrams ( Left ) and results of yeast two-hybrid analysis with deletion mutants ( Right ). Amino acid (aa) numbers at domain junctions are shown above schematics and amino acid residues contained in each deletion construct are indicated in clone name ( Lower Left ). Minimum critical regions necessary for interaction with binding partners are indicated by shaded bar. ( A ) Myozenin structure illustrating two putative α-helical domains (A1 and A2) and central glycine-rich domain (Gly). ( B ) α-Actinin-2 structure showing actin-binding domain (ABD), central repeats (R1–R4), spacer (S), and EF hand domain (EF). ( C ) γ-Filamin schematic illustrating ABD, repeats (numbered), repeats 19 and 20 interdomain insertion (I), and hinge region (H).
    Figure Legend Snippet: Schematic diagrams ( Left ) and results of yeast two-hybrid analysis with deletion mutants ( Right ). Amino acid (aa) numbers at domain junctions are shown above schematics and amino acid residues contained in each deletion construct are indicated in clone name ( Lower Left ). Minimum critical regions necessary for interaction with binding partners are indicated by shaded bar. ( A ) Myozenin structure illustrating two putative α-helical domains (A1 and A2) and central glycine-rich domain (Gly). ( B ) α-Actinin-2 structure showing actin-binding domain (ABD), central repeats (R1–R4), spacer (S), and EF hand domain (EF). ( C ) γ-Filamin schematic illustrating ABD, repeats (numbered), repeats 19 and 20 interdomain insertion (I), and hinge region (H).

    Techniques Used: Construct, Binding Assay

    21) Product Images from "Identification of novel sequence variations in microRNAs in chronic lymphocytic leukemia"

    Article Title: Identification of novel sequence variations in microRNAs in chronic lymphocytic leukemia

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgt396

    Frequency of minor alleles in CLL patients versus control population from the 1000 genomes project. The allelic frequencies of SNPs detected in miRNAs in CLL patients differed significantly ( P
    Figure Legend Snippet: Frequency of minor alleles in CLL patients versus control population from the 1000 genomes project. The allelic frequencies of SNPs detected in miRNAs in CLL patients differed significantly ( P

    Techniques Used:

    Novel 1 nt sequence variations detected in miRNAs. Five novel heterozygous variations, localized in various miRNA regions, were detected by resequencing microarray and confirmed by Sanger sequencing in five different CLL patients: ( A ) pri-miR-29a, ( B ) pre-miR-16-1, ( C ) pre-miR-372, ( D ) pre-miR-106b, ( E ) miR-142-3p. Variations localized downstream of the precursor hairpin, which contains the mature miRNA, are assigned ‘+’.
    Figure Legend Snippet: Novel 1 nt sequence variations detected in miRNAs. Five novel heterozygous variations, localized in various miRNA regions, were detected by resequencing microarray and confirmed by Sanger sequencing in five different CLL patients: ( A ) pri-miR-29a, ( B ) pre-miR-16-1, ( C ) pre-miR-372, ( D ) pre-miR-106b, ( E ) miR-142-3p. Variations localized downstream of the precursor hairpin, which contains the mature miRNA, are assigned ‘+’.

    Techniques Used: Sequencing, Microarray

    Secondary structures of wt miRNAs and miRNAs harboring sequence variations (novel variation/SNP) predicted by the RNAfold tool. ( A ) The novel variation found in pre-miR-16-1 (83G > C) had a dramatic effect on its secondary structure, whereas the known mutation present in its primary region (+7C > T) ( 9 ) had none. ( B ) pri-miR-29b-2 secondary structure was affected by polymorphic insertion (rs141961287: +107+A). Depicted are the most stable secondary structures with the lowest free energy as predicted by the RNAfold tool. Variations are designated in red and indicated by arrows. Mature miRNAs and miR-5p are designated in violet; mature miRNAs* and miR-3p are designated in blue. Pre-miRNA regions are designated in green; pri-miRNA regions are colorless.
    Figure Legend Snippet: Secondary structures of wt miRNAs and miRNAs harboring sequence variations (novel variation/SNP) predicted by the RNAfold tool. ( A ) The novel variation found in pre-miR-16-1 (83G > C) had a dramatic effect on its secondary structure, whereas the known mutation present in its primary region (+7C > T) ( 9 ) had none. ( B ) pri-miR-29b-2 secondary structure was affected by polymorphic insertion (rs141961287: +107+A). Depicted are the most stable secondary structures with the lowest free energy as predicted by the RNAfold tool. Variations are designated in red and indicated by arrows. Mature miRNAs and miR-5p are designated in violet; mature miRNAs* and miR-3p are designated in blue. Pre-miRNA regions are designated in green; pri-miRNA regions are colorless.

    Techniques Used: Sequencing, Mutagenesis

    22) Product Images from "Segmental Maternal UPD of Chromosome 7q in a Patient With Pendred and Silver Russell Syndromes-Like Features"

    Article Title: Segmental Maternal UPD of Chromosome 7q in a Patient With Pendred and Silver Russell Syndromes-Like Features

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2018.00600

    Clinical and molecular characterization of the case. (A) Frontal and lateral view of the proband at the age of 36 months. Note the mild frontal bossing, low-set posteriorly rotated ears, and thin lips. Earing aids are in place. The patient also displays brachydactyly of both hands and feet and clinodactily of V finger of hands. Other clinical findings are listed in the side table, where 3 out of 6 NH-CSS for SRS are in bold characters. (B) The LR-PCR amplicons of the patient (P) and both her father (F) and mother (M) were resolved on a 0.8% agarose gel. The patient (P) displayed a unique shorter band of about 2.5 kb in size, the father (F) showed a unique band of over 10 kb in size, corresponding to the expected 14.7 kb wild type band, whereas her mother (M) showed two bands corresponding to the wild type and the deleted alleles. MW, molecular weight. (C) The proximal and distal breakpoints of the SLC26A4 intragenic deletion were mapped within SLC26A4 IVS16 and the IVS20, respectively. The deletion is about 13 kb long. Sequence alignments of the junction fragments revealed an insertion of a part of CCDC126 IVS3. The rejoining between SLC26A4 IVS20 and CCDC126 IVS3 distal bkp occurred through a de novo 3 bp GCC insertion. (D) Sequencing of the RT-PCR amplicons, extending from exons 13–14 to 3′UTR of SLC26A4 , confirmed the homozygous deletion of exons 17–20 in the child (P). The father (F) showed only the long transcript corresponding to the wild type pendrin, whereas the mother (M) displayed a short transcript and a long one, consistent with a heterozygous state of the deletion. MW, molecular weight.
    Figure Legend Snippet: Clinical and molecular characterization of the case. (A) Frontal and lateral view of the proband at the age of 36 months. Note the mild frontal bossing, low-set posteriorly rotated ears, and thin lips. Earing aids are in place. The patient also displays brachydactyly of both hands and feet and clinodactily of V finger of hands. Other clinical findings are listed in the side table, where 3 out of 6 NH-CSS for SRS are in bold characters. (B) The LR-PCR amplicons of the patient (P) and both her father (F) and mother (M) were resolved on a 0.8% agarose gel. The patient (P) displayed a unique shorter band of about 2.5 kb in size, the father (F) showed a unique band of over 10 kb in size, corresponding to the expected 14.7 kb wild type band, whereas her mother (M) showed two bands corresponding to the wild type and the deleted alleles. MW, molecular weight. (C) The proximal and distal breakpoints of the SLC26A4 intragenic deletion were mapped within SLC26A4 IVS16 and the IVS20, respectively. The deletion is about 13 kb long. Sequence alignments of the junction fragments revealed an insertion of a part of CCDC126 IVS3. The rejoining between SLC26A4 IVS20 and CCDC126 IVS3 distal bkp occurred through a de novo 3 bp GCC insertion. (D) Sequencing of the RT-PCR amplicons, extending from exons 13–14 to 3′UTR of SLC26A4 , confirmed the homozygous deletion of exons 17–20 in the child (P). The father (F) showed only the long transcript corresponding to the wild type pendrin, whereas the mother (M) displayed a short transcript and a long one, consistent with a heterozygous state of the deletion. MW, molecular weight.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Sequencing, Reverse Transcription Polymerase Chain Reaction

    23) Product Images from "Identification of differentially expressed genes in isogenic highly metastatic and poorly metastatic cell lines of R3230AC rat mammary adenocarcinoma"

    Article Title: Identification of differentially expressed genes in isogenic highly metastatic and poorly metastatic cell lines of R3230AC rat mammary adenocarcinoma

    Journal: Cell Proliferation

    doi: 10.1046/j.1365-2184.2003.00289.x

    Differential screening of subtracted cDNAs. Randomly chosen bacterial colonies from forward‐ or reverse‐subtracted cDNA library were grown in 96‐well plate, amplified with PCR and arrayed on nylon membranes. (a) and (b) are identical membranes for forward‐subtracted clones; (c) and (d) for reverse‐subtracted clones. The membranes (a) and (d) were hybridized with a forward‐subtracted cDNA probe; the membranes (b) and (c) hybridized with a reverse‐subtracted cDNA probe. Wells number A‐1 and A‐4 of membrane (a) served as negative controls for hybridization (testis specific protein cDNA).
    Figure Legend Snippet: Differential screening of subtracted cDNAs. Randomly chosen bacterial colonies from forward‐ or reverse‐subtracted cDNA library were grown in 96‐well plate, amplified with PCR and arrayed on nylon membranes. (a) and (b) are identical membranes for forward‐subtracted clones; (c) and (d) for reverse‐subtracted clones. The membranes (a) and (d) were hybridized with a forward‐subtracted cDNA probe; the membranes (b) and (c) hybridized with a reverse‐subtracted cDNA probe. Wells number A‐1 and A‐4 of membrane (a) served as negative controls for hybridization (testis specific protein cDNA).

    Techniques Used: cDNA Library Assay, Amplification, Polymerase Chain Reaction, Clone Assay, Hybridization

    24) Product Images from "Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro"

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro

    Journal: Immunology

    doi: 10.1046/j.1365-2567.2003.01693.x

    Expression of cell surface molecules in lymph node-derived stromal cells. (a) Stromal cells derived from lymph nodes of IL-2Rβ −/− mice were stained with fluorescence-conjugated antibodies and were analysed by flow cytometry. Shaded peaks and open peaks with dotted lines indicate the fluorescence intensities of cells stained with a non-specific control antibody labelled with the same fluorescent reagents. (b) RNA was extracted from stromal cells derived from lymph nodes of IL-2Rβ −/− mice, and RT–PCR was performed using primers to detect the expression of indicated cytokines.
    Figure Legend Snippet: Expression of cell surface molecules in lymph node-derived stromal cells. (a) Stromal cells derived from lymph nodes of IL-2Rβ −/− mice were stained with fluorescence-conjugated antibodies and were analysed by flow cytometry. Shaded peaks and open peaks with dotted lines indicate the fluorescence intensities of cells stained with a non-specific control antibody labelled with the same fluorescent reagents. (b) RNA was extracted from stromal cells derived from lymph nodes of IL-2Rβ −/− mice, and RT–PCR was performed using primers to detect the expression of indicated cytokines.

    Techniques Used: Expressing, Derivative Assay, Mouse Assay, Staining, Fluorescence, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

    25) Product Images from "Characterization of a novel germline PALB2 duplication in a hereditary breast and ovarian cancer family"

    Article Title: Characterization of a novel germline PALB2 duplication in a hereditary breast and ovarian cancer family

    Journal: Breast cancer research and treatment

    doi: 10.1007/s10549-016-4021-7

    Breakpoint determination. a PALB2 exon 13 duplication confirmation by long-range PCR. An extra band of approximately 4–6 kb in size was detected in the proband’s blood DNA, which is absent in the negative control sample. b Localization of breakpoint. Electropherogram showing the breakpoint sequence. c Sequence alignment in the breakpoint region showing the 32-bp perfect identity between the intron 12 and 3′-UTR of PALB2
    Figure Legend Snippet: Breakpoint determination. a PALB2 exon 13 duplication confirmation by long-range PCR. An extra band of approximately 4–6 kb in size was detected in the proband’s blood DNA, which is absent in the negative control sample. b Localization of breakpoint. Electropherogram showing the breakpoint sequence. c Sequence alignment in the breakpoint region showing the 32-bp perfect identity between the intron 12 and 3′-UTR of PALB2

    Techniques Used: Polymerase Chain Reaction, Negative Control, Sequencing

    An Alu-mediated mechanism appears to be responsible for exon 13 duplication. A sequence analysis of the LR-PCR product allowed us to identify the duplication conjunctions which are located 1089 bp downstream of exon 12, in intron 12, and 989 bp downstream of the PALB2 3′-UTR
    Figure Legend Snippet: An Alu-mediated mechanism appears to be responsible for exon 13 duplication. A sequence analysis of the LR-PCR product allowed us to identify the duplication conjunctions which are located 1089 bp downstream of exon 12, in intron 12, and 989 bp downstream of the PALB2 3′-UTR

    Techniques Used: Sequencing, Polymerase Chain Reaction

    PALB2 Exon 13 duplication disrupts normal splicing and leads to frameshift. a RT-PCR products run on QIAxcel. An extra band was observed in the patient, patient’s 48-year-old sister, aunt, and 52-year-old sister. b Electropherogram showing the inserted sequence from the upper band in the patient of figure ( a ). The insertion leads to a new stop codon as indicated by the red box
    Figure Legend Snippet: PALB2 Exon 13 duplication disrupts normal splicing and leads to frameshift. a RT-PCR products run on QIAxcel. An extra band was observed in the patient, patient’s 48-year-old sister, aunt, and 52-year-old sister. b Electropherogram showing the inserted sequence from the upper band in the patient of figure ( a ). The insertion leads to a new stop codon as indicated by the red box

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing

    26) Product Images from "Determination of Enterococcus faecalis groESL Full-Length Sequence and Application for Species Identification"

    Article Title: Determination of Enterococcus faecalis groESL Full-Length Sequence and Application for Species Identification

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.39.9.3326-3331.2001

    Broad-range PCR-RFLP of groESL genes among eight enterococcal species. Lane M, DNA size markers. Lanes 1 to 8, Rsa I digestion; lanes 10 to 16, Hae III. Lanes 1 and 9, E. faecalis ; lanes 2 and 10, E. faecium ; lanes 3 and 11, E. casseliflavus ; lanes 4 and 12, E. gallinarum ; lanes 5 and 13, E. avium ; lanes 6 and 14, E. raffinosus ; lanes 7 and 15, E. durans ; and lanes 8 and 16, E. hirae .
    Figure Legend Snippet: Broad-range PCR-RFLP of groESL genes among eight enterococcal species. Lane M, DNA size markers. Lanes 1 to 8, Rsa I digestion; lanes 10 to 16, Hae III. Lanes 1 and 9, E. faecalis ; lanes 2 and 10, E. faecium ; lanes 3 and 11, E. casseliflavus ; lanes 4 and 12, E. gallinarum ; lanes 5 and 13, E. avium ; lanes 6 and 14, E. raffinosus ; lanes 7 and 15, E. durans ; and lanes 8 and 16, E. hirae .

    Techniques Used: Polymerase Chain Reaction

    27) Product Images from "Detection of virulence factors of South African Lactococcus garvieae isolated from rainbow trout, Oncorhynchus mykiss (Walbaum)"

    Article Title: Detection of virulence factors of South African Lactococcus garvieae isolated from rainbow trout, Oncorhynchus mykiss (Walbaum)

    Journal: The Onderstepoort Journal of Veterinary Research

    doi: 10.4102/ojvr.v85i1.1568

    Putative virulence factor genes were detected by polymerase chain reaction assays. Products are shown visualised on 1% agarose gels. The first lanes on all gels are size markers, and the subsequent lanes represent virulence genes in the following order: hly1 ; hly2 ; hly3 ; nox ; sod ; pavA ; psaA . (a), A1; (b), A2; (c), A3, (d), A5; (e), A6; (f), A11; (g), A12; (h), NCFB657.
    Figure Legend Snippet: Putative virulence factor genes were detected by polymerase chain reaction assays. Products are shown visualised on 1% agarose gels. The first lanes on all gels are size markers, and the subsequent lanes represent virulence genes in the following order: hly1 ; hly2 ; hly3 ; nox ; sod ; pavA ; psaA . (a), A1; (b), A2; (c), A3, (d), A5; (e), A6; (f), A11; (g), A12; (h), NCFB657.

    Techniques Used: Polymerase Chain Reaction

    28) Product Images from "Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products"

    Article Title: Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni111

    PCR of subdivided genomic sequences. Three regions of human genomic DNA (GenBank accession nos AC006454 , AC093734 and X91835 , with 5103, 5193 and 12 114 bp, respectively) were subdivided into nine 567, 577 and 1346 bp PCR sites, respectively. PCR was performed for each subdivided site using primer sets (20 bp each) corresponding to the terminal sequence of each site using the Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio). PCR was carried out in the absence and in the presence of Tth RecA protein and ATP. The products were electrophoresed and stained with ethidium bromide. ( a ) A diagrammatic representation of the subdivided region (5103 bp in GenBank accession no AC006454 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( b ) A diagrammatic representation of the subdivided region (5193 bp in GenBank accession no AC093734 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( c ) A diagrammatic representation of the subdivided region (12 114 bp in GenBank accession no X91835 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). Throughout (a–c), nine subdivided sites for each region are indicated as a-1 to a-9, b-1 to b-9 and c-1 to c-9. Nucleotide (nt) numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.
    Figure Legend Snippet: PCR of subdivided genomic sequences. Three regions of human genomic DNA (GenBank accession nos AC006454 , AC093734 and X91835 , with 5103, 5193 and 12 114 bp, respectively) were subdivided into nine 567, 577 and 1346 bp PCR sites, respectively. PCR was performed for each subdivided site using primer sets (20 bp each) corresponding to the terminal sequence of each site using the Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio). PCR was carried out in the absence and in the presence of Tth RecA protein and ATP. The products were electrophoresed and stained with ethidium bromide. ( a ) A diagrammatic representation of the subdivided region (5103 bp in GenBank accession no AC006454 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( b ) A diagrammatic representation of the subdivided region (5193 bp in GenBank accession no AC093734 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( c ) A diagrammatic representation of the subdivided region (12 114 bp in GenBank accession no X91835 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). Throughout (a–c), nine subdivided sites for each region are indicated as a-1 to a-9, b-1 to b-9 and c-1 to c-9. Nucleotide (nt) numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Techniques Used: Polymerase Chain Reaction, Genomic Sequencing, Sequencing, Staining

    PCR with primers carrying mismatched bases. PCR was performed at two human genomic sites with primers (20 bp), one of which (forward primer) carried one, two or three mismatched bases in the middle of the primer, in the absence (left) or presence (right) of Tth RecA protein and ATP using the Taq DNA polymerase ( rTaq DNA polymerase plus ‘hot start’ antibody). ( a ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 66 562 and nt 66 581 in GenBank accession no AC006454 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set a-1); lanes 2 and 6, PCR products using primers (primer set a-2 with one mismatched base at nt 66 566, T to C); lanes 3 and 7, PCR products using primers (primer set a-3 with two mismatched base at nt 66 566 and nt 66 571, both T to C); and lanes 4 and 8, PCR products using primers (primer set a-4 with three mismatched base at nt 66 566 and nt 66 571, T to C and nt 66 576, G to C). The oligonucleotide sequences used for the forward primers (mismatched bases are underlined) are as follows: primer set a-1, 5′-CATGGCACCTGCTCTGAGAC-3′; primer set a-2, 5′-CATGGCACC C GCTCTGAGAC-3′; primer set a-3, 5′-CATGGCACC C GCTC C GAGAC-3′; and primer set a-4, 5′-CATG C CACC C GCTC C GAGAC-3′. ( b ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 38 501 and nt 38 520 in GenBank accession no. AC0937734 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set b-1); lanes 2 and 6, PCR products using primers (primer set b-2 with one mismatched base at nt 38 505, G to A); lanes 3 and 7, PCR products using primers (primer set b-3 with two mismatched base at nt 38 505 and nt 38 510, both G to A); and lanes 4 and 8, PCR products using primers (primer set b-4 with three mismatched base at nt 38 505, nt 38 510 and nt 38 515, all G to A). The oligonucleotide sequences used for the forward primers are as follows: primer set b-1, 5′-ATCTGTGTGGTTCGGCTCTG-3′; primer set b-2, 5′-ATCTGTGTG A TTCGGCTCTG-3′; primer set b-3, 5′-ATCTGTGTG A TTCG A CTCTG-3′; and primer set b-4, 5′-ATCT A TGTG A TTCG A CTCTG-3′. ( c ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 63 957 and nt 63 976 in GenBank accession no. AC004975 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set c-1); lanes 2 and 6, PCR products using primers (primer set c-2 with one mismatched base at nt 63 961, A to T); lanes 3 and 7, PCR products using primers (primer set c-3 with two mismatched base at nt 63 961 and nt 63 966, A to T and C to T); and lanes 4 and 8, PCR products using primers (primer set c-4 with three mismatched base at nt 63 961, nt 63 966 and nt 63 971, A to T, C to T and G to T). The oligonucleotide sequences used for the forward primers are as follows: primer set c-1, 5′-GCAGGCACCAAGAACTACTG-3′; primer set c-2, 5′-GCAGGCACC T AGAACTACTG-3′; primer set c-3, 5′-GCAGGCACC T AGAA T TACTG-3′; and primer set c-4, 5′-GCAG T CACC T AGAA T TACTG-3′. The sequences for the backward primers are 5′-TCACCTCCCAGCCTGGCCCA-3′ for ( a ), 5′-AGGGAGATGTTCTCATAAAT-3′ and 5′-CTGTAAGTGGCAGACATTAC-3′ for ( b ). Nucleotide numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.
    Figure Legend Snippet: PCR with primers carrying mismatched bases. PCR was performed at two human genomic sites with primers (20 bp), one of which (forward primer) carried one, two or three mismatched bases in the middle of the primer, in the absence (left) or presence (right) of Tth RecA protein and ATP using the Taq DNA polymerase ( rTaq DNA polymerase plus ‘hot start’ antibody). ( a ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 66 562 and nt 66 581 in GenBank accession no AC006454 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set a-1); lanes 2 and 6, PCR products using primers (primer set a-2 with one mismatched base at nt 66 566, T to C); lanes 3 and 7, PCR products using primers (primer set a-3 with two mismatched base at nt 66 566 and nt 66 571, both T to C); and lanes 4 and 8, PCR products using primers (primer set a-4 with three mismatched base at nt 66 566 and nt 66 571, T to C and nt 66 576, G to C). The oligonucleotide sequences used for the forward primers (mismatched bases are underlined) are as follows: primer set a-1, 5′-CATGGCACCTGCTCTGAGAC-3′; primer set a-2, 5′-CATGGCACC C GCTCTGAGAC-3′; primer set a-3, 5′-CATGGCACC C GCTC C GAGAC-3′; and primer set a-4, 5′-CATG C CACC C GCTC C GAGAC-3′. ( b ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 38 501 and nt 38 520 in GenBank accession no. AC0937734 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set b-1); lanes 2 and 6, PCR products using primers (primer set b-2 with one mismatched base at nt 38 505, G to A); lanes 3 and 7, PCR products using primers (primer set b-3 with two mismatched base at nt 38 505 and nt 38 510, both G to A); and lanes 4 and 8, PCR products using primers (primer set b-4 with three mismatched base at nt 38 505, nt 38 510 and nt 38 515, all G to A). The oligonucleotide sequences used for the forward primers are as follows: primer set b-1, 5′-ATCTGTGTGGTTCGGCTCTG-3′; primer set b-2, 5′-ATCTGTGTG A TTCGGCTCTG-3′; primer set b-3, 5′-ATCTGTGTG A TTCG A CTCTG-3′; and primer set b-4, 5′-ATCT A TGTG A TTCG A CTCTG-3′. ( c ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 63 957 and nt 63 976 in GenBank accession no. AC004975 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set c-1); lanes 2 and 6, PCR products using primers (primer set c-2 with one mismatched base at nt 63 961, A to T); lanes 3 and 7, PCR products using primers (primer set c-3 with two mismatched base at nt 63 961 and nt 63 966, A to T and C to T); and lanes 4 and 8, PCR products using primers (primer set c-4 with three mismatched base at nt 63 961, nt 63 966 and nt 63 971, A to T, C to T and G to T). The oligonucleotide sequences used for the forward primers are as follows: primer set c-1, 5′-GCAGGCACCAAGAACTACTG-3′; primer set c-2, 5′-GCAGGCACC T AGAACTACTG-3′; primer set c-3, 5′-GCAGGCACC T AGAA T TACTG-3′; and primer set c-4, 5′-GCAG T CACC T AGAA T TACTG-3′. The sequences for the backward primers are 5′-TCACCTCCCAGCCTGGCCCA-3′ for ( a ), 5′-AGGGAGATGTTCTCATAAAT-3′ and 5′-CTGTAAGTGGCAGACATTAC-3′ for ( b ). Nucleotide numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Techniques Used: Polymerase Chain Reaction

    Effect of T.thermophilus RecA protein on PCR. PCR with Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio) for several randomly selected sequences (300–650 bp) in human genomic DNA was carried out in the absence or in the presence of the Tth RecA protein. ( a ) Control, PCR under the standard conditions described in Materials and Methods. ( b ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture). ( c ) Similar to (a), but with ATP (400 μM). ( d ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP (300 μM). ( e ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP-γS (300 μM). The products were electrophoresed and stained with ethidium bromide. Molecular weight markers are indicated on the right-hand side of these panels. The oligonucleotide sequences used for the primers were as follows: 5′-ACAATGGGCTCACTCACCCA-3′ and 5′-CTAAGACCAATGGATAGCTG-3′ for lane 1 (300 bp); 5′-GCTCAGCATGGTGGTGGCAT-3′ and 5′-CCTCATACCTTCCCCCCCAT-3′ for lane 2 (319 bp); 5′-GACTACTCTAGCGACTGTCC-3′ and 5′-GACAGCCACCAGATCCAATC-3′ for lane 3 (360 bp); 5′-AACCTCACAACCTTGGCTGA-3′ and 5′-TTCACAACTTAAGATTTGGC-3′ for lane 4 (400 bp); 5′-AGGCAACTAGGATGGTGTGG-3′ and 5′-CAGGGAGCGTGTCCATAGGG-3′ for lane 5 (450 bp); 5′-CTGCTGAAAGAGATGCGGTG-3′ and 5′-AGGAAAACAGCCCAAGGGAC-3′ for lane 6 (469 bp); and 5′-ACTTTGTTCTGAGCCTCACA-3′ and 5′-GTTGCCCAATCGCCCCTCTC-3′ for lane 7 (650 bp).
    Figure Legend Snippet: Effect of T.thermophilus RecA protein on PCR. PCR with Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio) for several randomly selected sequences (300–650 bp) in human genomic DNA was carried out in the absence or in the presence of the Tth RecA protein. ( a ) Control, PCR under the standard conditions described in Materials and Methods. ( b ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture). ( c ) Similar to (a), but with ATP (400 μM). ( d ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP (300 μM). ( e ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP-γS (300 μM). The products were electrophoresed and stained with ethidium bromide. Molecular weight markers are indicated on the right-hand side of these panels. The oligonucleotide sequences used for the primers were as follows: 5′-ACAATGGGCTCACTCACCCA-3′ and 5′-CTAAGACCAATGGATAGCTG-3′ for lane 1 (300 bp); 5′-GCTCAGCATGGTGGTGGCAT-3′ and 5′-CCTCATACCTTCCCCCCCAT-3′ for lane 2 (319 bp); 5′-GACTACTCTAGCGACTGTCC-3′ and 5′-GACAGCCACCAGATCCAATC-3′ for lane 3 (360 bp); 5′-AACCTCACAACCTTGGCTGA-3′ and 5′-TTCACAACTTAAGATTTGGC-3′ for lane 4 (400 bp); 5′-AGGCAACTAGGATGGTGTGG-3′ and 5′-CAGGGAGCGTGTCCATAGGG-3′ for lane 5 (450 bp); 5′-CTGCTGAAAGAGATGCGGTG-3′ and 5′-AGGAAAACAGCCCAAGGGAC-3′ for lane 6 (469 bp); and 5′-ACTTTGTTCTGAGCCTCACA-3′ and 5′-GTTGCCCAATCGCCCCTCTC-3′ for lane 7 (650 bp).

    Techniques Used: Polymerase Chain Reaction, Staining, Molecular Weight

    29) Product Images from "A Multiplex PCR Detection Assay for the Identification of Clinically Relevant Anaplasma Species in Field Blood Samples"

    Article Title: A Multiplex PCR Detection Assay for the Identification of Clinically Relevant Anaplasma Species in Field Blood Samples

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00606

    Optimization of the multiplex PCR components. (A) Annealing temperature gradients, M: DL2000 marker. Lanes 1–8: 64, 63, 62, 61, 60, 59, 58, and 57°C, respectively. (B) Dose of Anaplasma primers, lane M: DL2000 marker; Lanes 1–7: 0.08, 0.16, 0.24, 0.32, 0.4, 0.48, and 0.56 μM, respectively; Lane N: negative control. (C) La Taq DNA polymerase. M: DL2000 marker; Lanes 1–7: 0.75, 1.0, 1.25, 1.5, 1.75, 2, and 2.25 U, respectively; Lane N: negative control.
    Figure Legend Snippet: Optimization of the multiplex PCR components. (A) Annealing temperature gradients, M: DL2000 marker. Lanes 1–8: 64, 63, 62, 61, 60, 59, 58, and 57°C, respectively. (B) Dose of Anaplasma primers, lane M: DL2000 marker; Lanes 1–7: 0.08, 0.16, 0.24, 0.32, 0.4, 0.48, and 0.56 μM, respectively; Lane N: negative control. (C) La Taq DNA polymerase. M: DL2000 marker; Lanes 1–7: 0.75, 1.0, 1.25, 1.5, 1.75, 2, and 2.25 U, respectively; Lane N: negative control.

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Marker, Negative Control

    30) Product Images from "Serine Protease PRSS23 Is Upregulated by Estrogen Receptor ? and Associated with Proliferation of Breast Cancer Cells"

    Article Title: Serine Protease PRSS23 Is Upregulated by Estrogen Receptor ? and Associated with Proliferation of Breast Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030397

    Expression analyses of PRSS23 in human cell lines. Expression levels of PRSS23 as well as ERα were analyzed in eight different cell lines: MCF-7, BT-474, Hs.578t, MDA-MB-231, T-47D (all breast cancer), MCF-10A (mammary epithelial), RL95-2 (endometrial cancer), and Ca-SKi (cervical cancer) cell lines. A. Immunoblot analysis showed protein expression level of ERα in these human cell lines. B. qRT-PCR analysis showed relative gene expression of PRSS23 mRNA level. C. Immunoblot analysis showed protein expression level of PRSS23 and GAPDH in these human cell lines. The cell lysate was loaded 20 µg protein for each well in immunoblot anaylsis. qRT-PCR was performed in duplicate.
    Figure Legend Snippet: Expression analyses of PRSS23 in human cell lines. Expression levels of PRSS23 as well as ERα were analyzed in eight different cell lines: MCF-7, BT-474, Hs.578t, MDA-MB-231, T-47D (all breast cancer), MCF-10A (mammary epithelial), RL95-2 (endometrial cancer), and Ca-SKi (cervical cancer) cell lines. A. Immunoblot analysis showed protein expression level of ERα in these human cell lines. B. qRT-PCR analysis showed relative gene expression of PRSS23 mRNA level. C. Immunoblot analysis showed protein expression level of PRSS23 and GAPDH in these human cell lines. The cell lysate was loaded 20 µg protein for each well in immunoblot anaylsis. qRT-PCR was performed in duplicate.

    Techniques Used: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR

    E 2 -activated ERα enhances PRSS23 expression in MCF-7 cells. A. MCF-7 cells were treated with 1 nM E 2 , 25 ppm ethanol, 5 µM Tam, and 0.5% dimethyl sulfoxide (DMSO) in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. The bar plots depicted the results of time-lapse profiling of PRSS23 mRNA levels at 6, 12, and 24 h. All experiments were performed in triplicate. The bars represent relative expression levels of PRSS23 after treatment, which was normalized to the level of 6 h-treated cells (mean ± S.E.M.). B. MDA-MB-231 cells were treated with 1 nM E 2 in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. Expression of PRSS23 (upper panel) and pS2 (lower panel) was evaluated by qRT-PCR at 0, 6, 12, and 24 h. The bars represented the gene expression levels of PRSS23 after treatment, which was normalized to the level of untreated cells (mean ± S.E.M.).
    Figure Legend Snippet: E 2 -activated ERα enhances PRSS23 expression in MCF-7 cells. A. MCF-7 cells were treated with 1 nM E 2 , 25 ppm ethanol, 5 µM Tam, and 0.5% dimethyl sulfoxide (DMSO) in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. The bar plots depicted the results of time-lapse profiling of PRSS23 mRNA levels at 6, 12, and 24 h. All experiments were performed in triplicate. The bars represent relative expression levels of PRSS23 after treatment, which was normalized to the level of 6 h-treated cells (mean ± S.E.M.). B. MDA-MB-231 cells were treated with 1 nM E 2 in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. Expression of PRSS23 (upper panel) and pS2 (lower panel) was evaluated by qRT-PCR at 0, 6, 12, and 24 h. The bars represented the gene expression levels of PRSS23 after treatment, which was normalized to the level of untreated cells (mean ± S.E.M.).

    Techniques Used: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR

    31) Product Images from "Viability of an Escherichia coli pgsA Null Mutant Lacking Detectable Phosphatidylglycerol and Cardiolipin"

    Article Title: Viability of an Escherichia coli pgsA Null Mutant Lacking Detectable Phosphatidylglycerol and Cardiolipin

    Journal: Journal of Bacteriology

    doi:

    PCR analysis of the pgsA alleles of E. coli pgsA mutants. PCR products amplified with Ex Taq DNA polymerase were subjected to 1.2% agarose gel electrophoresis. Lanes 1, W3110; lanes 2, S301; lanes 3, MDL12; lanes 4 to 6, independent clones of the transductants (S330). Primer pairs FPPG5-ASFPPG1 (a) and FPPG5-ASFPPG3 (b) were used. The design and sequences of the primers are described in Materials and Methods. With the former primer pair, the wild-type pgsA allele and the pgsA :: kan allele gave products of 0.71 and 1.9 kbp, respectively. With the latter primer pair, the wild-type pgsA and the pgsA :: kan alleles gave products of 0.5 and 1.7 kbp, respectively. A DNA fragment of ca. 1.5 kbp which appeared in MDL12 (panel b, lane 3) may be the product of a false annealing of the antisense primer with a site in lacZ ′ fused to pgsA ). The molecular size markers included (two left lanes of each gel) were λ- Hin dIII digest (23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kbp) and λ- Eco T14 I digest (19.3, 7.7, 6.2, 4.3, 3.5, 2.7, 1.9, 1.5, 0.93, and 0.42 kbp).
    Figure Legend Snippet: PCR analysis of the pgsA alleles of E. coli pgsA mutants. PCR products amplified with Ex Taq DNA polymerase were subjected to 1.2% agarose gel electrophoresis. Lanes 1, W3110; lanes 2, S301; lanes 3, MDL12; lanes 4 to 6, independent clones of the transductants (S330). Primer pairs FPPG5-ASFPPG1 (a) and FPPG5-ASFPPG3 (b) were used. The design and sequences of the primers are described in Materials and Methods. With the former primer pair, the wild-type pgsA allele and the pgsA :: kan allele gave products of 0.71 and 1.9 kbp, respectively. With the latter primer pair, the wild-type pgsA and the pgsA :: kan alleles gave products of 0.5 and 1.7 kbp, respectively. A DNA fragment of ca. 1.5 kbp which appeared in MDL12 (panel b, lane 3) may be the product of a false annealing of the antisense primer with a site in lacZ ′ fused to pgsA ). The molecular size markers included (two left lanes of each gel) were λ- Hin dIII digest (23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kbp) and λ- Eco T14 I digest (19.3, 7.7, 6.2, 4.3, 3.5, 2.7, 1.9, 1.5, 0.93, and 0.42 kbp).

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Clone Assay

    32) Product Images from "The 23S rRNA gene PCR-RFLP used for characterization of porcine intestinal spirochete isolates"

    Article Title: The 23S rRNA gene PCR-RFLP used for characterization of porcine intestinal spirochete isolates

    Journal: Journal of Veterinary Science

    doi: 10.4142/jvs.2006.7.3.277

    PCR-RFLP fragments of the 23S rRNA gene in 3% agarose gel electrophoresis digestion with Taq I. Lane M: 100 bp DNA ladder; lane 1: B. hyodysenteriae B204; lane 2: B. hyodysenteriae B234; lane 3: B. hyodysenteriae B169; lane 4: B. pilosicoli P43/6/78; lane 5 to 14: B. hyodysenteriae field isolates; lane 15: B. murdochii 56-150; lane 16: B. intermedia PWS/A; lane 17: B. innocens B256.
    Figure Legend Snippet: PCR-RFLP fragments of the 23S rRNA gene in 3% agarose gel electrophoresis digestion with Taq I. Lane M: 100 bp DNA ladder; lane 1: B. hyodysenteriae B204; lane 2: B. hyodysenteriae B234; lane 3: B. hyodysenteriae B169; lane 4: B. pilosicoli P43/6/78; lane 5 to 14: B. hyodysenteriae field isolates; lane 15: B. murdochii 56-150; lane 16: B. intermedia PWS/A; lane 17: B. innocens B256.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    33) Product Images from "The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity"

    Article Title: The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082149

    Knockdown of the pancreas acinar cell Zip5 gene has no effect on the rapid accumulation of zinc but apparently impairs zinc retention in the pancreas. (A) Control littermates and pancreas-specific Zip5 -knockout ( Zip5 Panc KO ) mice (n = 4– 5) were killed 8 days after the last tamoxifen injection. Intestine, pancreas and liver were harvested from mice fed normal chow (ZnA) or normal chow plus 250 ppm zinc in the drinking water (ZnE) during those 8 days and pancreatic zinc was quantified using ICP-MS and is expressed as ppm/dry weight of tissue. (B) Control and Zip5 Panc KO mice were injected I.P. with 6.25 mg 67 Zn/kg body weight and the pancreas was harvested at 0 h, 2 h and 4 hr after the injection or (C) 27 hr after the injection. The ratio of 67 Zn/ 66 Zn was measured by ICP-MS. The natural ratio of these stable isotopes is 0.146. Only pancreatic zinc is shown since no other significant changes in any other element were found (*; P
    Figure Legend Snippet: Knockdown of the pancreas acinar cell Zip5 gene has no effect on the rapid accumulation of zinc but apparently impairs zinc retention in the pancreas. (A) Control littermates and pancreas-specific Zip5 -knockout ( Zip5 Panc KO ) mice (n = 4– 5) were killed 8 days after the last tamoxifen injection. Intestine, pancreas and liver were harvested from mice fed normal chow (ZnA) or normal chow plus 250 ppm zinc in the drinking water (ZnE) during those 8 days and pancreatic zinc was quantified using ICP-MS and is expressed as ppm/dry weight of tissue. (B) Control and Zip5 Panc KO mice were injected I.P. with 6.25 mg 67 Zn/kg body weight and the pancreas was harvested at 0 h, 2 h and 4 hr after the injection or (C) 27 hr after the injection. The ratio of 67 Zn/ 66 Zn was measured by ICP-MS. The natural ratio of these stable isotopes is 0.146. Only pancreatic zinc is shown since no other significant changes in any other element were found (*; P

    Techniques Used: Knock-Out, Mouse Assay, Injection, Mass Spectrometry

    Knockout of the pancreas acinar cell Zip5 gene is mosaic. Newly weaned mice were injected with tamoxifen to induce Zip5 recombination in the pancreas by activation of Cre-ERT2 expressed from an Ela-Cre-ERT2 transgene. ( A ) Northern blot detection of Zip5 mRNA in the pancreas of control ( Control ) littermates and pancreas-specific Zip5 -knockout ( Zip 5 Panc KO ) mice killed 5 days after the last tamoxifen injection. ( B ) Detection of ZIP5 protein using immunohistochemistry ( ZIP5 IHC ). Paraffin sections of pancreas were incubated with an anti-ZIP5 peptide antibody and specific binding was detected as a dark brown precipitate on the baso-lateral surfaces of acinar cells. Sections from 2 control mice and 3 different knockout mice are shown at 200× magnification. Quantification of the number of ZIP5 immuno-positive and negative cells per field of view in multiple sections from several mice indicated an efficiency of the knockout of ∼70 to ∼90%. In Part B number 1 knockout, the percentage of remaining positive cells was 8% (6/80) indicating a 92% efficacy of the knockout. In part B number 2 knockout, the percentage of remaining positive cells 32% (39/120) indicating an ∼68% efficacy of the knockout. I ; indicates Islets of Langerhans: D ; indicates a pancreatic duct.
    Figure Legend Snippet: Knockout of the pancreas acinar cell Zip5 gene is mosaic. Newly weaned mice were injected with tamoxifen to induce Zip5 recombination in the pancreas by activation of Cre-ERT2 expressed from an Ela-Cre-ERT2 transgene. ( A ) Northern blot detection of Zip5 mRNA in the pancreas of control ( Control ) littermates and pancreas-specific Zip5 -knockout ( Zip 5 Panc KO ) mice killed 5 days after the last tamoxifen injection. ( B ) Detection of ZIP5 protein using immunohistochemistry ( ZIP5 IHC ). Paraffin sections of pancreas were incubated with an anti-ZIP5 peptide antibody and specific binding was detected as a dark brown precipitate on the baso-lateral surfaces of acinar cells. Sections from 2 control mice and 3 different knockout mice are shown at 200× magnification. Quantification of the number of ZIP5 immuno-positive and negative cells per field of view in multiple sections from several mice indicated an efficiency of the knockout of ∼70 to ∼90%. In Part B number 1 knockout, the percentage of remaining positive cells was 8% (6/80) indicating a 92% efficacy of the knockout. In part B number 2 knockout, the percentage of remaining positive cells 32% (39/120) indicating an ∼68% efficacy of the knockout. I ; indicates Islets of Langerhans: D ; indicates a pancreatic duct.

    Techniques Used: Knock-Out, Mouse Assay, Injection, Activation Assay, Northern Blot, Immunohistochemistry, Incubation, Binding Assay

    Structures of the pre- and post-Cre floxed mouse Zip5 gene and integration and genotyping screen designs. ( A ) The mouse Zip5 gene was captured using gap-repair and manipulated using galK recombineering. Exons ( 1–12 ) and the exon encoding transmembrane domain 1 ( TMD1 ) are indicated, as are the positions of LoxP sites (intron 4 and downstream of PGK Neo ), the PGK-neomycin ( PGK Neo ) cassette and the locations of primers used for genotyping. The LoxP site in intron 4 is flanked by an EcoRV restriction enzyme cleavage site. ( B ) The structure of the Zip5 gene after Cre recombination is shown. Recombination eliminates the transmembrane domain of ZIP5. ( C ) The floxed Zip5 gene was targeted into E14 ES cells and properly targeted ES cells were identified by long range PCR using flanking and internal primers. PCR products from the wild-type ( Wt ) and floxed ( Fx ) alleles are indicated. EcoRV cleavage was used to differentiate between the floxed and wild-type alleles in the 5′ PCR screen whereas the 3′ PCR screen yielded the predicted larger product from the wild-type allele. Targeted ES cells were used to generate mice homozygous for the floxed Zip5 allele. ( D ) Mice were genotyped by PCR amplification of the intron 4 region containing the LoxP site. The PCR product from homozygous Zip5 floxed mice before Cre-induced recombination is shown in the left lane while that from control mice is shown in the center lane and that from Zip5 -knockout mice ( Ko ) is shown in the right lane . For the intestine- and pancreas-specific knockout mice, detection of Zip5 mRNA and/or protein was employed to monitor the efficacy of recombination.
    Figure Legend Snippet: Structures of the pre- and post-Cre floxed mouse Zip5 gene and integration and genotyping screen designs. ( A ) The mouse Zip5 gene was captured using gap-repair and manipulated using galK recombineering. Exons ( 1–12 ) and the exon encoding transmembrane domain 1 ( TMD1 ) are indicated, as are the positions of LoxP sites (intron 4 and downstream of PGK Neo ), the PGK-neomycin ( PGK Neo ) cassette and the locations of primers used for genotyping. The LoxP site in intron 4 is flanked by an EcoRV restriction enzyme cleavage site. ( B ) The structure of the Zip5 gene after Cre recombination is shown. Recombination eliminates the transmembrane domain of ZIP5. ( C ) The floxed Zip5 gene was targeted into E14 ES cells and properly targeted ES cells were identified by long range PCR using flanking and internal primers. PCR products from the wild-type ( Wt ) and floxed ( Fx ) alleles are indicated. EcoRV cleavage was used to differentiate between the floxed and wild-type alleles in the 5′ PCR screen whereas the 3′ PCR screen yielded the predicted larger product from the wild-type allele. Targeted ES cells were used to generate mice homozygous for the floxed Zip5 allele. ( D ) Mice were genotyped by PCR amplification of the intron 4 region containing the LoxP site. The PCR product from homozygous Zip5 floxed mice before Cre-induced recombination is shown in the left lane while that from control mice is shown in the center lane and that from Zip5 -knockout mice ( Ko ) is shown in the right lane . For the intestine- and pancreas-specific knockout mice, detection of Zip5 mRNA and/or protein was employed to monitor the efficacy of recombination.

    Techniques Used: Polymerase Chain Reaction, Mouse Assay, Amplification, Knock-Out

    Knockdown of the pancreas acinar cell Zip5 gene sensitizes mice to zinc-induced acute pancreatitis. Two weeks after the last tamoxifen injection, control (Control) littermates and pancreas-specific Zip5 -knockout ( Zip5 Panc KO ) (n = 5) were given an I.P. injection of zinc sulfate (12.5 mg zinc/kg body weight) and 48 hr later pancreata were harvested and paraffin sections were prepared and stained with hematoxylin and eosin (panels A–D) or stained for ZIP5 using immunohistochemistry (panels E and F). Panels A and B represent control mice whereas panels C and D represent ZIP5- knockout mice ( Zip5 Panc KO ). Dark brown deposits on the basolateral surfaces of acinar cells indicate positive staining. Black arrowheads in panel F demarcate large cytoplasmic vaculoles found in acinar cells of Zip5 Panc KO mice in response to zinc. Panels A–D are photographed at 200× magnification and panels E and F are photographed at 1000× magnification.
    Figure Legend Snippet: Knockdown of the pancreas acinar cell Zip5 gene sensitizes mice to zinc-induced acute pancreatitis. Two weeks after the last tamoxifen injection, control (Control) littermates and pancreas-specific Zip5 -knockout ( Zip5 Panc KO ) (n = 5) were given an I.P. injection of zinc sulfate (12.5 mg zinc/kg body weight) and 48 hr later pancreata were harvested and paraffin sections were prepared and stained with hematoxylin and eosin (panels A–D) or stained for ZIP5 using immunohistochemistry (panels E and F). Panels A and B represent control mice whereas panels C and D represent ZIP5- knockout mice ( Zip5 Panc KO ). Dark brown deposits on the basolateral surfaces of acinar cells indicate positive staining. Black arrowheads in panel F demarcate large cytoplasmic vaculoles found in acinar cells of Zip5 Panc KO mice in response to zinc. Panels A–D are photographed at 200× magnification and panels E and F are photographed at 1000× magnification.

    Techniques Used: Mouse Assay, Injection, Knock-Out, Staining, Immunohistochemistry

    Detection of α-amylase in zinc-induced large cytoplasmic vaculoles in acinar cells of Zip5 Panc KO mice. Two weeks after the last tamoxifen injection pancreas-specific Zip5 -knockout ( Zip5 Panc KO ) mice were given an I.P. injection of zinc sulfate (12.5 mg zinc/kg body weight) and 24 hr later pancreata were harvested and paraffin sections were prepared and stained for α-amylase using immunohistochemistry. Sections from two different mice are shown. Dark brown deposits in acinar cells indicate positive staining. Black arrowheads demarcate large cytoplasmic vacuoles containing α-amylase. Sections were photographed at 630× magnification. I ; indicates an Islet of Langerhans.
    Figure Legend Snippet: Detection of α-amylase in zinc-induced large cytoplasmic vaculoles in acinar cells of Zip5 Panc KO mice. Two weeks after the last tamoxifen injection pancreas-specific Zip5 -knockout ( Zip5 Panc KO ) mice were given an I.P. injection of zinc sulfate (12.5 mg zinc/kg body weight) and 24 hr later pancreata were harvested and paraffin sections were prepared and stained for α-amylase using immunohistochemistry. Sections from two different mice are shown. Dark brown deposits in acinar cells indicate positive staining. Black arrowheads demarcate large cytoplasmic vacuoles containing α-amylase. Sections were photographed at 630× magnification. I ; indicates an Islet of Langerhans.

    Techniques Used: Mouse Assay, Injection, Knock-Out, Staining, Immunohistochemistry

    Loss of function of the intestine Zip5 gene results in the accumulation of zinc in the pancreas. Newly weaned mice were injected with tamoxifen to induce Zip5 recombination in the intestine by activation of Cre-ERT2 expressed from a vil-Cre-ERT2 transgene. ( A ) Northern blot detection of Zip5 and Zip4 mRNAs in the small intestine of control ( Con ) littermates and intestine-specific Zip5 -knockout ( Zip5 Intest KO ) mice. Results from 2 mice per group are shown but 5 mice per group were analyzed with identical results. ( B ) Intestine, pancreas and liver were harvested from control and Zip5 Intest KO mice fed normal chow ( ZnA ) or normal chow plus 250 ppm zinc in the drinking water ( ZnE ) for 8 days after knocking out the Zip5 gene in the intestine. Tissue elements were quantified using ICP-MS and are expressed as ppm/dry weight of tissue. Only zinc levels in the pancreas are shown because this was the only change detected (n = 5: ****; P
    Figure Legend Snippet: Loss of function of the intestine Zip5 gene results in the accumulation of zinc in the pancreas. Newly weaned mice were injected with tamoxifen to induce Zip5 recombination in the intestine by activation of Cre-ERT2 expressed from a vil-Cre-ERT2 transgene. ( A ) Northern blot detection of Zip5 and Zip4 mRNAs in the small intestine of control ( Con ) littermates and intestine-specific Zip5 -knockout ( Zip5 Intest KO ) mice. Results from 2 mice per group are shown but 5 mice per group were analyzed with identical results. ( B ) Intestine, pancreas and liver were harvested from control and Zip5 Intest KO mice fed normal chow ( ZnA ) or normal chow plus 250 ppm zinc in the drinking water ( ZnE ) for 8 days after knocking out the Zip5 gene in the intestine. Tissue elements were quantified using ICP-MS and are expressed as ppm/dry weight of tissue. Only zinc levels in the pancreas are shown because this was the only change detected (n = 5: ****; P

    Techniques Used: Mouse Assay, Injection, Activation Assay, Northern Blot, Knock-Out, Mass Spectrometry

    Total knockout of the Zip5 gene results in increased hepatic zinc and modestly attenuates pancreatic zinc accumulation. Mice with homozygous floxed Zip5 genes were crossed with mice that ubiquitously express CRE recombinase under control of the EIIa promoter. Offspring with complete recombination of the Zip5 gene in tail DNA were identified and crossed to create a colony of Zip5 - knockout mice ( Zip5 Total KO ). Zip5 Fx/Fx mice served as controls (Con). (A) Mice (n = 4 or 5) were maintained on normal diet (ZnA) diet and pancreas (Panc), liver and intestine (Intest) were harvested two weeks after weaning and from age matched Zip5 Total KO and control and tissue elements were quantified using ICP-MS and are expressed as ppm/dry weight. Zinc in the liver was the only element which differed significantly between the Zip5 Total KO and control mice (P = 0.0032). (B) Intestine, pancreas and liver were harvested from control and Zip5 Total KO mice fed normal chow (ZnA) or normal chow plus 250 ppm zinc in the drinking water (ZnE) for 8 days and tissue elements were quantified using ICP-MS. Only pancreatic zinc is shown since no other significant changes in any other element were found (**; P
    Figure Legend Snippet: Total knockout of the Zip5 gene results in increased hepatic zinc and modestly attenuates pancreatic zinc accumulation. Mice with homozygous floxed Zip5 genes were crossed with mice that ubiquitously express CRE recombinase under control of the EIIa promoter. Offspring with complete recombination of the Zip5 gene in tail DNA were identified and crossed to create a colony of Zip5 - knockout mice ( Zip5 Total KO ). Zip5 Fx/Fx mice served as controls (Con). (A) Mice (n = 4 or 5) were maintained on normal diet (ZnA) diet and pancreas (Panc), liver and intestine (Intest) were harvested two weeks after weaning and from age matched Zip5 Total KO and control and tissue elements were quantified using ICP-MS and are expressed as ppm/dry weight. Zinc in the liver was the only element which differed significantly between the Zip5 Total KO and control mice (P = 0.0032). (B) Intestine, pancreas and liver were harvested from control and Zip5 Total KO mice fed normal chow (ZnA) or normal chow plus 250 ppm zinc in the drinking water (ZnE) for 8 days and tissue elements were quantified using ICP-MS. Only pancreatic zinc is shown since no other significant changes in any other element were found (**; P

    Techniques Used: Knock-Out, Mouse Assay, Mass Spectrometry

    34) Product Images from "The DFR locus: A smart landing pad for targeted transgene insertion in tomato"

    Article Title: The DFR locus: A smart landing pad for targeted transgene insertion in tomato

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208395

    Targeted gene insertion at the dfr locus. (A) Schematic representation of HDR-mediated gene insertion at the dfr mutant locus using two sgRNAs: sgRNA-DFR#3 and sgRNA-DFR#4 targeting the dfr deletion junction and exon 6 (E6 yellow box). The PAM site is in green, the target sequence in red and the sgRNA in blue. The promoter and terminator sequences are represented with pale blue and orange boxes respectively (P and Ter). Gene targeting (GT) homologous sequences are shown with boxes with a hatched pattern. The NptII gene insertion is represented by a blue box. The primers used for detection and sequencing are shown as dark blue arrows. (B) Regenerating explants on kanamycin selective media after Agrobacterium transformation of the T2 plant DFR64a with a single binary vector carrying two sgRNAs and a DNA repair template containing the DFR sequence and NptII gene. The purple coloured events can be visually identified (red arrow) (C) Purple coloured plantlets regenerating on kanamycin-containing media (D) In vitro regenerated T0 plants with HDR-mediated-gene targeting in the DFR landing pad isolated in a tube. (E) PCR analysis for the detection of the precise HR with the repair template at the 5’ junction, 3’ junction and the presence of the NptII gene with primers GT1F and GT1R in six T0 plantlets which showed anthocyanin pigmentation. WT: wild-type; Neg. control: negative control green plantlet; dfr DFR64a: deleted dfr mutant used for the HDR-mediated experiment; 183, 387, 463, 161, 303 and 524: in vitro samples from T0 plantlets issued from targeted insertion events visually screened for their anthocyanin pigmentation. (F) Long range PCR with primers GT3F and GT3R on two different targeted insertions. WT: wild-type; 463 and 524: samples from T0 plantlets from event number 463 and number 524 (with anthocyanin pigmentation). (G) Sequencing of the 5’ and 3’ junctions at the DFR locus in T0 plantlets with recovered anthocyanin pigmentation for events 161, 183, 303, 387, 463 and 524. The sequence of the 3’ junction was not obtained for event number 183. Coloured lower-case letters indicate the end and the beginning of the homologous sequence used in the DNA donor template. Black upper-case letters show the genomic sequences surrounding the insertion. Mutations in the sequences compared to expected sequence are shown with yellow boxes and bold letters.
    Figure Legend Snippet: Targeted gene insertion at the dfr locus. (A) Schematic representation of HDR-mediated gene insertion at the dfr mutant locus using two sgRNAs: sgRNA-DFR#3 and sgRNA-DFR#4 targeting the dfr deletion junction and exon 6 (E6 yellow box). The PAM site is in green, the target sequence in red and the sgRNA in blue. The promoter and terminator sequences are represented with pale blue and orange boxes respectively (P and Ter). Gene targeting (GT) homologous sequences are shown with boxes with a hatched pattern. The NptII gene insertion is represented by a blue box. The primers used for detection and sequencing are shown as dark blue arrows. (B) Regenerating explants on kanamycin selective media after Agrobacterium transformation of the T2 plant DFR64a with a single binary vector carrying two sgRNAs and a DNA repair template containing the DFR sequence and NptII gene. The purple coloured events can be visually identified (red arrow) (C) Purple coloured plantlets regenerating on kanamycin-containing media (D) In vitro regenerated T0 plants with HDR-mediated-gene targeting in the DFR landing pad isolated in a tube. (E) PCR analysis for the detection of the precise HR with the repair template at the 5’ junction, 3’ junction and the presence of the NptII gene with primers GT1F and GT1R in six T0 plantlets which showed anthocyanin pigmentation. WT: wild-type; Neg. control: negative control green plantlet; dfr DFR64a: deleted dfr mutant used for the HDR-mediated experiment; 183, 387, 463, 161, 303 and 524: in vitro samples from T0 plantlets issued from targeted insertion events visually screened for their anthocyanin pigmentation. (F) Long range PCR with primers GT3F and GT3R on two different targeted insertions. WT: wild-type; 463 and 524: samples from T0 plantlets from event number 463 and number 524 (with anthocyanin pigmentation). (G) Sequencing of the 5’ and 3’ junctions at the DFR locus in T0 plantlets with recovered anthocyanin pigmentation for events 161, 183, 303, 387, 463 and 524. The sequence of the 3’ junction was not obtained for event number 183. Coloured lower-case letters indicate the end and the beginning of the homologous sequence used in the DNA donor template. Black upper-case letters show the genomic sequences surrounding the insertion. Mutations in the sequences compared to expected sequence are shown with yellow boxes and bold letters.

    Techniques Used: Mutagenesis, Sequencing, Transformation Assay, Plasmid Preparation, In Vitro, Isolation, Polymerase Chain Reaction, Negative Control, Genomic Sequencing

    35) Product Images from "Evolution of MIR168 paralogs in Brassicaceae"

    Article Title: Evolution of MIR168 paralogs in Brassicaceae

    Journal: BMC Evolutionary Biology

    doi: 10.1186/1471-2148-9-62

    Phylogenetic reconstruction of MIR168 in Brassicaeae . Phylogenetic reconstruction of MIR168a and MIR168b in the Brassicaceae family compared with a phylogenetic tree drawn using the IT S and EIF3 markers. Values at the branch roots correspond to majority rule consensus bootstrap values ≥ 50%. A) ITS-EIF3 phylogenetic tree; B) MIR168a-MIR168b phylogenetic tree.
    Figure Legend Snippet: Phylogenetic reconstruction of MIR168 in Brassicaeae . Phylogenetic reconstruction of MIR168a and MIR168b in the Brassicaceae family compared with a phylogenetic tree drawn using the IT S and EIF3 markers. Values at the branch roots correspond to majority rule consensus bootstrap values ≥ 50%. A) ITS-EIF3 phylogenetic tree; B) MIR168a-MIR168b phylogenetic tree.

    Techniques Used:

    Expression pattern of MIR168 paralogs in Arabidopsis . A) Genomic region encompassing MIR168a ; B) genomic region encompassing MIR168b . Black box: mature miR168; dashed box: miR168*; white boxes: 20 bp sequences forming the basal stem; light gray box: miR168 loop region; dark gray boxes: nearest exons in the genes upstream and downstream of MIR168 , arrows indicate gene orientation. Distances are drawn to scale, with the exception of pre-miR168 (to a larger scale for clarity); +1 is the first nucleotide of the mature miR168. The pMIR168a::GFP-GUS , pMIR168b1::GFP-GUS and pMIR168b2::GFP-GUS constructs are represented underneath the genomic regions. C) GUS-staining of Arabidopsis transformant lines carrying the pMIR168a::GFP-GUS and pMIR168b1::GFP-GUS constructs.
    Figure Legend Snippet: Expression pattern of MIR168 paralogs in Arabidopsis . A) Genomic region encompassing MIR168a ; B) genomic region encompassing MIR168b . Black box: mature miR168; dashed box: miR168*; white boxes: 20 bp sequences forming the basal stem; light gray box: miR168 loop region; dark gray boxes: nearest exons in the genes upstream and downstream of MIR168 , arrows indicate gene orientation. Distances are drawn to scale, with the exception of pre-miR168 (to a larger scale for clarity); +1 is the first nucleotide of the mature miR168. The pMIR168a::GFP-GUS , pMIR168b1::GFP-GUS and pMIR168b2::GFP-GUS constructs are represented underneath the genomic regions. C) GUS-staining of Arabidopsis transformant lines carrying the pMIR168a::GFP-GUS and pMIR168b1::GFP-GUS constructs.

    Techniques Used: Expressing, Construct, Staining

    Synteny conservation and duplication dating of MIR168 paralogs . A) Synteny conservation of the genomic regions encompassing MIR168a and MIR168b in A. thaliana and P. trichocarpa . Arrows represent MIR168a and MIR168b ; the squares represent coding genes with at least one homolog in both genomes; the black lines represent RBMs and the gray lines connect BLASTP hits with lower homology within the same syntenic regions. Dashed lines connect At4g19410 homologs; diagonal lines on Ptc_LG_III represent a 7 Mbp long region not syntenic to Arabidopsis. B) Phylogenetic reconstruction of At4g19410 homologs in the Arabidopsis and poplar genomes. The portion of the linearized tree representing the homologs of At4g19410 located in the same genomic regions as MIR168a and MIR168b is highlighted in black. Values at the branch roots correspond to majority rule consensus bootstrap values ≥ 50%. Ath: A. thaliana ; Ptc: P. trichocarpa ; Ks: number of synonymous nucleotide substitutions per synonymous site.
    Figure Legend Snippet: Synteny conservation and duplication dating of MIR168 paralogs . A) Synteny conservation of the genomic regions encompassing MIR168a and MIR168b in A. thaliana and P. trichocarpa . Arrows represent MIR168a and MIR168b ; the squares represent coding genes with at least one homolog in both genomes; the black lines represent RBMs and the gray lines connect BLASTP hits with lower homology within the same syntenic regions. Dashed lines connect At4g19410 homologs; diagonal lines on Ptc_LG_III represent a 7 Mbp long region not syntenic to Arabidopsis. B) Phylogenetic reconstruction of At4g19410 homologs in the Arabidopsis and poplar genomes. The portion of the linearized tree representing the homologs of At4g19410 located in the same genomic regions as MIR168a and MIR168b is highlighted in black. Values at the branch roots correspond to majority rule consensus bootstrap values ≥ 50%. Ath: A. thaliana ; Ptc: P. trichocarpa ; Ks: number of synonymous nucleotide substitutions per synonymous site.

    Techniques Used:

    Stem-loop structure and conservation of the pre-miR168 homologs . LOGO representation of the stem-loop structure of the pre-miR168 homologs in Brassicaceae species. The base composition is indicated at each position. Gray lines correspond to the pre-microRNA processing sites. A) pre-miR168a; B) pre-miR168b.
    Figure Legend Snippet: Stem-loop structure and conservation of the pre-miR168 homologs . LOGO representation of the stem-loop structure of the pre-miR168 homologs in Brassicaceae species. The base composition is indicated at each position. Gray lines correspond to the pre-microRNA processing sites. A) pre-miR168a; B) pre-miR168b.

    Techniques Used:

    Thermodynamic stability and nucleotide substitution profiles of pre-miR168a and pre-miR168b . A) Thermodynamic stability profile of pre-miR168a and pre-miR168b in the Brassicaceae family. Free energy values are given in kcal/mole. Vertical bars: between-species variability calculated as double standard error. B) Distribution of nucleotide substitutions with respect to base pairing in the pre-miR168a and pre-miR168b secondary structures. Yellow: structurally conservative base substitution; ochre: base substitution comporting a change in length of a bulge loop; blue: base substitution comporting a change from unpaired to paired bases; red: base substitution comporting a change from paired to unpaired bases. The rate of nucleotide substitution is given in percentages.
    Figure Legend Snippet: Thermodynamic stability and nucleotide substitution profiles of pre-miR168a and pre-miR168b . A) Thermodynamic stability profile of pre-miR168a and pre-miR168b in the Brassicaceae family. Free energy values are given in kcal/mole. Vertical bars: between-species variability calculated as double standard error. B) Distribution of nucleotide substitutions with respect to base pairing in the pre-miR168a and pre-miR168b secondary structures. Yellow: structurally conservative base substitution; ochre: base substitution comporting a change in length of a bulge loop; blue: base substitution comporting a change from unpaired to paired bases; red: base substitution comporting a change from paired to unpaired bases. The rate of nucleotide substitution is given in percentages.

    Techniques Used:

    36) Product Images from "Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia"

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    Journal: Microbiology and Immunology

    doi: 10.1111/1348-0421.12648

    Estimated CTX prophage region structure of V. cholerae strain M25, the representative of group ET‐8. Profiles of CTX prophage region‐specific RFLP and PCR of strain M25 are unique and it was categorized as an independent group, ET‐8. The best estimated model for CTX prophage region of strain M25 is “TLC–RS1–CTX‐1–RS1–RTX” on chromosome I and there are no CTX prophage‐associated genes on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain M25, the representative of group ET‐8. Profiles of CTX prophage region‐specific RFLP and PCR of strain M25 are unique and it was categorized as an independent group, ET‐8. The best estimated model for CTX prophage region of strain M25 is “TLC–RS1–CTX‐1–RS1–RTX” on chromosome I and there are no CTX prophage‐associated genes on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C7, the representative of group ET‐6. Profiles of CTX prophage region‐specific RFLP and PCR of strain C7 are unique and it was categorized as an independent group, ET‐6. The best estimated model for CTX prophage region of strain C7 is “TLC– + CTX‐1– # CTX‐1– + CTX‐1– + CTX‐1–RTX” on chromosome I; no CTX prophage‐associated genes are present on chromosome II. + CTX‐1, CTX‐1 harboring SNPs in rstA (G301A), rstB (T84C), and in ctxA (G622A); # CTX‐1, CTX‐1 harboring SNPs in rstB (T84C) and in ctxA (G622A).
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain C7, the representative of group ET‐6. Profiles of CTX prophage region‐specific RFLP and PCR of strain C7 are unique and it was categorized as an independent group, ET‐6. The best estimated model for CTX prophage region of strain C7 is “TLC– + CTX‐1– # CTX‐1– + CTX‐1– + CTX‐1–RTX” on chromosome I; no CTX prophage‐associated genes are present on chromosome II. + CTX‐1, CTX‐1 harboring SNPs in rstA (G301A), rstB (T84C), and in ctxA (G622A); # CTX‐1, CTX‐1 harboring SNPs in rstB (T84C) and in ctxA (G622A).

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain P2, the representative of group ET‐5. Four V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐5. The strain P2 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain P2, the representative of group ET‐5. Four V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐5. The strain P2 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C1, the representative of group ET‐3. Profiles of CTX prophage region‐specific RFLP and PCR of strain C1 are unique and it was categorized as an independent group, ET‐3. The best estimated model for CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain C1, the representative of group ET‐3. Profiles of CTX prophage region‐specific RFLP and PCR of strain C1 are unique and it was categorized as an independent group, ET‐3. The best estimated model for CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain J6, the representative of group ET‐2. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR and were categorized into a group designated as ET‐2. The strain J6 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain J6, the representative of group ET‐2. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR and were categorized into a group designated as ET‐2. The strain J6 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C2, the representative of group ET‐4. Profiles of CTX prophage region‐specific RFLP and PCR of strain C2 are unique and it was categorized as an independent group, ET‐4. The best estimated model for CTX prophage region of strain C2 is “TLC–CTX‐1–RS1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and a single “VCET1_GI” on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain C2, the representative of group ET‐4. Profiles of CTX prophage region‐specific RFLP and PCR of strain C2 are unique and it was categorized as an independent group, ET‐4. The best estimated model for CTX prophage region of strain C2 is “TLC–CTX‐1–RS1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and a single “VCET1_GI” on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    Estimated CTX prophage region structure of Vibrio cholerae strain 341, the representative of group ET‐1. Six V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐1. (a) The strain 341 was chosen as a representative and sequenced. The best estimated model, “TLC–*RS1–CTX‐1–VCET1_GI–VCET1_GI–RTX” for chromosome I and “VCET1_GI” for chromosome II, is shown. The sites to which each of zot , ctxA and rstC probes bind and produced fragments in the RFLP analysis are shown in the figure. (b) Estimated structure of a novel genomic island VCET1_GI. Structures of satellite phage RS1 and CTX‐1 are shown in references. TLC, toxin‐linked cryptic gene cluster; CTX‐1, CTX prophage harboring rstR El Tor and ctxB3 ; *RS1, RS1 which harbors five nucleotide substitutions in rstA compared with that of N16961.
    Figure Legend Snippet: Estimated CTX prophage region structure of Vibrio cholerae strain 341, the representative of group ET‐1. Six V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐1. (a) The strain 341 was chosen as a representative and sequenced. The best estimated model, “TLC–*RS1–CTX‐1–VCET1_GI–VCET1_GI–RTX” for chromosome I and “VCET1_GI” for chromosome II, is shown. The sites to which each of zot , ctxA and rstC probes bind and produced fragments in the RFLP analysis are shown in the figure. (b) Estimated structure of a novel genomic island VCET1_GI. Structures of satellite phage RS1 and CTX‐1 are shown in references. TLC, toxin‐linked cryptic gene cluster; CTX‐1, CTX prophage harboring rstR El Tor and ctxB3 ; *RS1, RS1 which harbors five nucleotide substitutions in rstA compared with that of N16961.

    Techniques Used: Polymerase Chain Reaction, Produced, Thin Layer Chromatography

    Estimated CTX prophage region structure of V. cholerae strain P16, the representative of group ET‐7. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐7. The strain P16 was chosen as a representative and sequenced. The best estimated model for CTX prophage region of strain P16 is “TLC–CTX‐1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and “VCET1_GI–VCET1_GI” on chromosome II.
    Figure Legend Snippet: Estimated CTX prophage region structure of V. cholerae strain P16, the representative of group ET‐7. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐7. The strain P16 was chosen as a representative and sequenced. The best estimated model for CTX prophage region of strain P16 is “TLC–CTX‐1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and “VCET1_GI–VCET1_GI” on chromosome II.

    Techniques Used: Polymerase Chain Reaction

    37) Product Images from "Normal Development and Fertility of Knockout Mice Lacking the Tumor Suppressor Gene LRP1b Suggest Functional Compensation by LRP1"

    Article Title: Normal Development and Fertility of Knockout Mice Lacking the Tumor Suppressor Gene LRP1b Suggest Functional Compensation by LRP1

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.9.3782-3793.2004

    Tissue specific alternative splicing of the cytoplasmic LRP1b tail. (A) Total RNA from mouse brain, adrenal gland, testis, and liver was transcribed by reverse transcriptase and subsequently amplified by PCR using primers P1 and P2 (upper panel, a), primers P3 and P2 (middle panel, b), or by primers specific for β-actin as a control (lower panel). The size of the amplification products with the LRP1b specific primers is shown. (B) The location of primers P1, P2, and P3 within the cytoplasmic tail of LRP1b is shown.
    Figure Legend Snippet: Tissue specific alternative splicing of the cytoplasmic LRP1b tail. (A) Total RNA from mouse brain, adrenal gland, testis, and liver was transcribed by reverse transcriptase and subsequently amplified by PCR using primers P1 and P2 (upper panel, a), primers P3 and P2 (middle panel, b), or by primers specific for β-actin as a control (lower panel). The size of the amplification products with the LRP1b specific primers is shown. (B) The location of primers P1, P2, and P3 within the cytoplasmic tail of LRP1b is shown.

    Techniques Used: Amplification, Polymerase Chain Reaction

    Localization of LRP1b expression in the brain. Sagittal brain sections from adult wild-type mice were hybridized with antisense probes specific for LRP1b (A to C), PSD-95 (D to F), and AIP (G to I) as well as a sense control probe (J to L) by in situ hybridization. For each probe, a 1.6× objective magnification of the total brain as well as 4× objective magnifications of the hippocampus and the region around the fourth ventricle are shown. Scale bar, 1 mm.
    Figure Legend Snippet: Localization of LRP1b expression in the brain. Sagittal brain sections from adult wild-type mice were hybridized with antisense probes specific for LRP1b (A to C), PSD-95 (D to F), and AIP (G to I) as well as a sense control probe (J to L) by in situ hybridization. For each probe, a 1.6× objective magnification of the total brain as well as 4× objective magnifications of the hippocampus and the region around the fourth ventricle are shown. Scale bar, 1 mm.

    Techniques Used: Expressing, Mouse Assay, In Situ Hybridization

    Genotyping and absence of LRP1b protein expression in LRP1b −/− mice. (A) Genomic DNA was prepared from the tails of wild-type mice (lane 1) and mice heterozygous (lane 2) and homozygous (lane 3) for the disruption of the LRP1b gene. The DNA was amplified by PCR with allele-specific primers to detect the wild-type LRP1b allele (500 bp) and disrupted allele (300 bp), respectively. (B) Genomic DNA from the tails of mice with the genotypes indicated was digested with BamHI and BglII and separated on a 0.6% agarose gel. Southern blotting was performed with a 32 P-labeled probe 3′ of the short arm homology region. The size of the bands is shown (5-kb wild-type allele, 6.5-kb disrupted allele). (C) Crude membrane fractions were prepared from the brains of three homozygous LRP1b −/− mice (lanes 1 to 3) and three wild-type controls (lanes 4 to 6). Equal amounts of total protein (100 μg) were separated by SDS-4% PAGE, transferred to nitrocellulose membranes and subjected to Western blotting using antibodies against the carboxyl terminus of LRP1b (upper panel) and LRP1 (lower panel).
    Figure Legend Snippet: Genotyping and absence of LRP1b protein expression in LRP1b −/− mice. (A) Genomic DNA was prepared from the tails of wild-type mice (lane 1) and mice heterozygous (lane 2) and homozygous (lane 3) for the disruption of the LRP1b gene. The DNA was amplified by PCR with allele-specific primers to detect the wild-type LRP1b allele (500 bp) and disrupted allele (300 bp), respectively. (B) Genomic DNA from the tails of mice with the genotypes indicated was digested with BamHI and BglII and separated on a 0.6% agarose gel. Southern blotting was performed with a 32 P-labeled probe 3′ of the short arm homology region. The size of the bands is shown (5-kb wild-type allele, 6.5-kb disrupted allele). (C) Crude membrane fractions were prepared from the brains of three homozygous LRP1b −/− mice (lanes 1 to 3) and three wild-type controls (lanes 4 to 6). Equal amounts of total protein (100 μg) were separated by SDS-4% PAGE, transferred to nitrocellulose membranes and subjected to Western blotting using antibodies against the carboxyl terminus of LRP1b (upper panel) and LRP1 (lower panel).

    Techniques Used: Expressing, Mouse Assay, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Southern Blot, Labeling, Polyacrylamide Gel Electrophoresis, Western Blot

    Expression of LRP1b ectodomains and binding to RAP. (A) HEK 293 cells were transfected with plasmids coding for ectodomains containing the ligand-binding domain region I, II, III, or IV of LRP1b with three amino-terminal FLAG tags. At 48 h after transfection, the supernatants were harvested and loaded directly or after pull-down with GST-RAP or GST on SDS gels (4 to 15% acrylamide). After transfer to nitrocellulose membranes, the ectodomains were detected with a monoclonal anti-FLAG antibody. (B) The structures of the FLAG-tagged LRP1b ectodomains are shown.
    Figure Legend Snippet: Expression of LRP1b ectodomains and binding to RAP. (A) HEK 293 cells were transfected with plasmids coding for ectodomains containing the ligand-binding domain region I, II, III, or IV of LRP1b with three amino-terminal FLAG tags. At 48 h after transfection, the supernatants were harvested and loaded directly or after pull-down with GST-RAP or GST on SDS gels (4 to 15% acrylamide). After transfer to nitrocellulose membranes, the ectodomains were detected with a monoclonal anti-FLAG antibody. (B) The structures of the FLAG-tagged LRP1b ectodomains are shown.

    Techniques Used: Expressing, Binding Assay, Transfection, Ligand Binding Assay

    Knockout strategy for the targeted inactivation of the LRP1b gene in the mouse. A targeting vector was constructed to replace exon 88 of the LRP1b gene, which codes for the membrane-spanning segment, with a neo cassette. Exons are symbolized as numbered rectangles. The position of the primers used for genotyping by PCR is indicated by arrowheads. The black horizontal bar indicates the location of the probe used for Southern blotting. The expected sizes of the bands for the wild-type and disrupted allele after digestion with BamHI and BglII are shown.
    Figure Legend Snippet: Knockout strategy for the targeted inactivation of the LRP1b gene in the mouse. A targeting vector was constructed to replace exon 88 of the LRP1b gene, which codes for the membrane-spanning segment, with a neo cassette. Exons are symbolized as numbered rectangles. The position of the primers used for genotyping by PCR is indicated by arrowheads. The black horizontal bar indicates the location of the probe used for Southern blotting. The expected sizes of the bands for the wild-type and disrupted allele after digestion with BamHI and BglII are shown.

    Techniques Used: Knock-Out, Plasmid Preparation, Construct, Polymerase Chain Reaction, Southern Blot

    Synaptic transmission and plasticity in mice lacking LRP1b and/or RAP. Hippocampal slices were prepared from the brains of wild-type (WT) mice, RAP −/− mice, LRP1b −/− mice, and LRP1b −/− ; RAP −/− double knockout mice. Baseline synaptic transmission represented as the slope of the field excitatory postsynaptic potential (fEPSP) compared to fiber volley amplitude at increasing stimulus intensities (A); PPF at IPI of 20, 50, 100, 200, and 300 ms (B); and LTP induced with two trains of 100-Hz stimulation for 1 s separated with a 20-s interval (indicated by an arrow) (C) are shown for each genotype. Data are shown as means ± standard errors of the means (error bars).
    Figure Legend Snippet: Synaptic transmission and plasticity in mice lacking LRP1b and/or RAP. Hippocampal slices were prepared from the brains of wild-type (WT) mice, RAP −/− mice, LRP1b −/− mice, and LRP1b −/− ; RAP −/− double knockout mice. Baseline synaptic transmission represented as the slope of the field excitatory postsynaptic potential (fEPSP) compared to fiber volley amplitude at increasing stimulus intensities (A); PPF at IPI of 20, 50, 100, 200, and 300 ms (B); and LTP induced with two trains of 100-Hz stimulation for 1 s separated with a 20-s interval (indicated by an arrow) (C) are shown for each genotype. Data are shown as means ± standard errors of the means (error bars).

    Techniques Used: Transmission Assay, Mouse Assay, Double Knockout, Mass Spectrometry

    Tissue distribution of LRP1b in the mouse. (A) Total RNA was isolated from murine tissues and amplified by RT-PCR using primers specific for the cytoplasmic tail of LRP1b. The size of the observed fragments (400 and 300 bp) is indicated. RNA integrity was ascertained by control amplifications with primers specific for mouse β-actin. (B) Crude membrane preparations of different mouse tissues were prepared from wild-type mice. Total protein (100 μg per lane) was separated by SDS-PAGE (4 to 15% acrylamide), transferred to nitrocellulose membranes and subjected to Western blotting using the affinity-purified LRP1b antibody.
    Figure Legend Snippet: Tissue distribution of LRP1b in the mouse. (A) Total RNA was isolated from murine tissues and amplified by RT-PCR using primers specific for the cytoplasmic tail of LRP1b. The size of the observed fragments (400 and 300 bp) is indicated. RNA integrity was ascertained by control amplifications with primers specific for mouse β-actin. (B) Crude membrane preparations of different mouse tissues were prepared from wild-type mice. Total protein (100 μg per lane) was separated by SDS-PAGE (4 to 15% acrylamide), transferred to nitrocellulose membranes and subjected to Western blotting using the affinity-purified LRP1b antibody.

    Techniques Used: Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, SDS Page, Western Blot, Affinity Purification

    LRP1b is not processed by furin. (A) Crude membrane preparations from mouse kidney (10 μg, lane 1), liver (50 μg, lane 2) and brain (100 μg, lane 3 wild-type, lane 4 LRP1b −/− ) were separated by SDS-PAGE (4 to 15% acrylamide) and subjected to Western blotting with antibodies against megalin (lane 1), LRP1 (lane 2) and LRP1b (lanes 3 and 4). The arrowhead indicates the expected position of a carboxyl-terminal fragment after furin cleavage, which is prominent in the case of LRP1 but missing in the other two receptors. (B) HEK 293 cells were transfected with membrane-bound minireceptors containing ligand-binding domain region IV of LRP1 (lane 1), region II of LRP1 (lane 2), region IV of LRP1b (lane 3), and region IV of LRP1b with an N-terminal FLAG tag (lane 5). Lane 4 is a control from mock-transfected cells. Equal amounts of lysates from these cells were separated by PAGE (4 to 15% acrylamide) followed by Western blotting with antibodies against the carboxyl terminus of LRP1 (lane 1 and 2), LRP1b (Lane 3 and 4), or against the FLAG epitope (lane 5). The arrowhead indicates the position of an expected carboxyl-terminal fragment after furin processing, which is only seen in cells transfected with the region IV LRP1 minireceptor. (C) Crude membrane preparations from mouse heart (lanes 1 to 4) or lysates from HEK 293 cells transfected with the LRP1b minireceptor were left untreated (lanes 1 and 5), or treated with endoglycosidase H (lanes 2 and 6), neuraminidase (lanes 3, and 7), or PNGase F (lanes 4 and 8). After overnight incubation, SDS-6% PAGE was performed followed by Western blotting using the anti-VLDL receptor (lanes 1 to 4) and anti-LRP1b (lanes 5 to 8) antibodies, respectively. (D) The structure of the LRP1b minireceptor with and without the amino-terminal FLAG tag is shown.
    Figure Legend Snippet: LRP1b is not processed by furin. (A) Crude membrane preparations from mouse kidney (10 μg, lane 1), liver (50 μg, lane 2) and brain (100 μg, lane 3 wild-type, lane 4 LRP1b −/− ) were separated by SDS-PAGE (4 to 15% acrylamide) and subjected to Western blotting with antibodies against megalin (lane 1), LRP1 (lane 2) and LRP1b (lanes 3 and 4). The arrowhead indicates the expected position of a carboxyl-terminal fragment after furin cleavage, which is prominent in the case of LRP1 but missing in the other two receptors. (B) HEK 293 cells were transfected with membrane-bound minireceptors containing ligand-binding domain region IV of LRP1 (lane 1), region II of LRP1 (lane 2), region IV of LRP1b (lane 3), and region IV of LRP1b with an N-terminal FLAG tag (lane 5). Lane 4 is a control from mock-transfected cells. Equal amounts of lysates from these cells were separated by PAGE (4 to 15% acrylamide) followed by Western blotting with antibodies against the carboxyl terminus of LRP1 (lane 1 and 2), LRP1b (Lane 3 and 4), or against the FLAG epitope (lane 5). The arrowhead indicates the position of an expected carboxyl-terminal fragment after furin processing, which is only seen in cells transfected with the region IV LRP1 minireceptor. (C) Crude membrane preparations from mouse heart (lanes 1 to 4) or lysates from HEK 293 cells transfected with the LRP1b minireceptor were left untreated (lanes 1 and 5), or treated with endoglycosidase H (lanes 2 and 6), neuraminidase (lanes 3, and 7), or PNGase F (lanes 4 and 8). After overnight incubation, SDS-6% PAGE was performed followed by Western blotting using the anti-VLDL receptor (lanes 1 to 4) and anti-LRP1b (lanes 5 to 8) antibodies, respectively. (D) The structure of the LRP1b minireceptor with and without the amino-terminal FLAG tag is shown.

    Techniques Used: SDS Page, Western Blot, Transfection, Ligand Binding Assay, FLAG-tag, Polyacrylamide Gel Electrophoresis, Incubation

    38) Product Images from "Potential Spermatogenesis Recovery with Bone Marrow Mesenchymal Stem Cells in an Azoospermic Rat Model"

    Article Title: Potential Spermatogenesis Recovery with Bone Marrow Mesenchymal Stem Cells in an Azoospermic Rat Model

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms150813151

    Relative level of spermatogenic specific genes expression in recipient rat testicular tissue after BMSCs transplantation detected by real time PCR. Vasa, dazl, smad1 and stella expression were detected 2 weeks post-transplantation and increased to a relatively high level at 4 to 8 weeks. C-kit and GCNF were expressed at relatively high levels immediately after injection, and expression levels decreased to almost zero within 2 weeks. GCNF expression increased gradually at 4 weeks post-transplantation, while expression of c-kit remained at a relatively low level.
    Figure Legend Snippet: Relative level of spermatogenic specific genes expression in recipient rat testicular tissue after BMSCs transplantation detected by real time PCR. Vasa, dazl, smad1 and stella expression were detected 2 weeks post-transplantation and increased to a relatively high level at 4 to 8 weeks. C-kit and GCNF were expressed at relatively high levels immediately after injection, and expression levels decreased to almost zero within 2 weeks. GCNF expression increased gradually at 4 weeks post-transplantation, while expression of c-kit remained at a relatively low level.

    Techniques Used: Expressing, Transplantation Assay, Real-time Polymerase Chain Reaction, Injection

    39) Product Images from "AID assists DNMT1 to attenuate BCL6 expression through DNA methylation in diffuse large B-cell lymphoma cell lines"

    Article Title: AID assists DNMT1 to attenuate BCL6 expression through DNA methylation in diffuse large B-cell lymphoma cell lines

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2020.01.002

    Model for AID-DNMT1 complex suppressing BCL6 expression in DLBCL. (A) Loss of AID or DNMT1 causes instability of AID-DNMT1 complex. The disassociation of AID-DNMT1 from BCL6 promoter induces demethylation of BCL6 promoter, thus induces increased BCL6 expression in DLBCL. (B) AID-DNMT1 complex binds to the −0.4 kb −0 kb region of BCL6 promoter, their cooperation maintains methylation of BCL6 promoter and inhibits BCL6 expression in DLBCL. (C) MG132 treatment stabilizes AID-DNMT1 complex to bind to BCL6 promoter, drives dynamic methylation of BCL6 promoter and down regulates BCL6 , which shows a treatment of MG132 to BCL6-driven DLBCL.
    Figure Legend Snippet: Model for AID-DNMT1 complex suppressing BCL6 expression in DLBCL. (A) Loss of AID or DNMT1 causes instability of AID-DNMT1 complex. The disassociation of AID-DNMT1 from BCL6 promoter induces demethylation of BCL6 promoter, thus induces increased BCL6 expression in DLBCL. (B) AID-DNMT1 complex binds to the −0.4 kb −0 kb region of BCL6 promoter, their cooperation maintains methylation of BCL6 promoter and inhibits BCL6 expression in DLBCL. (C) MG132 treatment stabilizes AID-DNMT1 complex to bind to BCL6 promoter, drives dynamic methylation of BCL6 promoter and down regulates BCL6 , which shows a treatment of MG132 to BCL6-driven DLBCL.

    Techniques Used: Expressing, Methylation

    The stabilized AID-DNMT1 complex suppresses BCL6 expression in DLBCL cells. (A) Immunoblot measurement of DNMT1 and AID protein level was performed in 4WT and 4AIDKO, 10WT and 10AIDKO, 19WT and 19AIDKO cells, respectively. GAPDH protein was used as an internal control. (B) DNMT3A, DNMT3B and AID proteins were measured in WT and AIDKO DLBCL cells by immunoblots. GAPDH protein was taken as an internal control. (C) Immunoblot detection of DNMT1, AID and BCL6 proteins was performed in SU-DHL-4 cells with CRISPR/Cas9 including three gRNAs for depleting DNMT1. DNMT1KO3 was the gRNA with the best efficiency in DNMT1 knock out. GAPDH protein was used as an internal control. (D) DNMT1, AID and BCL6 protein levels were detected in 10WT, 19WT and 10DNMT1KO, 19DNMT1KO cells by immunoblots, and GAPDH protein was used as an internal control. (E) BCL6 transcripts were detected in 4WT and 4AIDKO cells after 5-Azacytidine (10 μM) (5-Aza) treatment for 24 hours in vitro by qRT-PCR. Data shown are representative of 3 technical replicates. (F) DNMT1, BCL6 and AID protein levels were detected in 4WT and 4AIDKO, 10WT and 10AIDKO, 19WT and 19AIDKO cells treated with 5-Azacytidine as in E. GAPDH protein was used as an internal control. (G) DNMT1 transcripts were detected in 4WT and 4AIDKO, 10WT and 10AIDKO, 19WT and 19AIDKO cells by qRT-PCR. Data shown are representative of 3 technical replicates. (H) Immunoblots of DNMT1 and AID protein levels were performed in 4, 10 and 19 cells treated with MG132 (10 μM) and/or 5-Azacytidine (10 μM). GAPDH protein was taken as an internal control. The 5-Azacytidine treatment was 24 hours, and MG132 treatment was 8 hours, the combined treatment of DLBCL cells (4, 10 and 19) was performed by adding MG132 following 5-Azacytidine treatment for 16 hours to continuously treat for 8 hours. (I, J) DNMT1 and AID proteins were detected by immunoblots after immunoprecipitation (IP) by anti-AID pulldown (I) and anti-DNMT1 (J) in 4, 10 and 19 cells. Data shown are representative of 3 independent experiments. Data are presented as mean ± SD. *,** and *** represent p
    Figure Legend Snippet: The stabilized AID-DNMT1 complex suppresses BCL6 expression in DLBCL cells. (A) Immunoblot measurement of DNMT1 and AID protein level was performed in 4WT and 4AIDKO, 10WT and 10AIDKO, 19WT and 19AIDKO cells, respectively. GAPDH protein was used as an internal control. (B) DNMT3A, DNMT3B and AID proteins were measured in WT and AIDKO DLBCL cells by immunoblots. GAPDH protein was taken as an internal control. (C) Immunoblot detection of DNMT1, AID and BCL6 proteins was performed in SU-DHL-4 cells with CRISPR/Cas9 including three gRNAs for depleting DNMT1. DNMT1KO3 was the gRNA with the best efficiency in DNMT1 knock out. GAPDH protein was used as an internal control. (D) DNMT1, AID and BCL6 protein levels were detected in 10WT, 19WT and 10DNMT1KO, 19DNMT1KO cells by immunoblots, and GAPDH protein was used as an internal control. (E) BCL6 transcripts were detected in 4WT and 4AIDKO cells after 5-Azacytidine (10 μM) (5-Aza) treatment for 24 hours in vitro by qRT-PCR. Data shown are representative of 3 technical replicates. (F) DNMT1, BCL6 and AID protein levels were detected in 4WT and 4AIDKO, 10WT and 10AIDKO, 19WT and 19AIDKO cells treated with 5-Azacytidine as in E. GAPDH protein was used as an internal control. (G) DNMT1 transcripts were detected in 4WT and 4AIDKO, 10WT and 10AIDKO, 19WT and 19AIDKO cells by qRT-PCR. Data shown are representative of 3 technical replicates. (H) Immunoblots of DNMT1 and AID protein levels were performed in 4, 10 and 19 cells treated with MG132 (10 μM) and/or 5-Azacytidine (10 μM). GAPDH protein was taken as an internal control. The 5-Azacytidine treatment was 24 hours, and MG132 treatment was 8 hours, the combined treatment of DLBCL cells (4, 10 and 19) was performed by adding MG132 following 5-Azacytidine treatment for 16 hours to continuously treat for 8 hours. (I, J) DNMT1 and AID proteins were detected by immunoblots after immunoprecipitation (IP) by anti-AID pulldown (I) and anti-DNMT1 (J) in 4, 10 and 19 cells. Data shown are representative of 3 independent experiments. Data are presented as mean ± SD. *,** and *** represent p

    Techniques Used: Expressing, Western Blot, CRISPR, Knock-Out, In Vitro, Quantitative RT-PCR, Immunoprecipitation

    The deamination of AID has no role for DNA demethylation on BCL6 gene. (A) BCL6 transcripts were detected in 4WT and 4AIDKO, 10WT and 10AIDKO, 19WT and 19AIDKO by qRT-PCR. Data shown are representative of 3 technical replicates. (B) BCL6 protein levels were measured in 4WT and 4AIDKO, 10AIDKO and 19AIDKO cells by immunoblots, and GAPDH protein was taken as an internal control. (C) Schematic diagram shows AID mediated BCL6 mutation region. Not drawn to scale. (D) Histograms represent point mutation frequency of BCL6 promoter in 4WT and 4AIDKO cells. Mutation was measured in DNA from 2 independent preparations. (E) Schematic diagram shows CpG sites in the promoter of BCL6 gene. The hollow box represents BCL6 promoter. The shadow boxes represent CpG sites. The segment for 17 CpG sites locates in −1.07 kb to −0.84 kb of BCL6 promoter. The segment for 4 CpG sites is in the −0.51 kb to −0.24 kb of BCL6 promoter. Not drawn to scale. The bisulfite sequencing was used to examine methylation status of these CpG sites in BCL6 promoter. Black circles signify methylated CpGs, and white circles indicate demethylated CpGs. (F) Histograms indicate results for demethylation frequency of CpG sites in BCL6 promoter as detecting in E. Methylation was measured in DNA from 2 independent preparations. Data are presented as mean ± SD. *,** and *** represent p
    Figure Legend Snippet: The deamination of AID has no role for DNA demethylation on BCL6 gene. (A) BCL6 transcripts were detected in 4WT and 4AIDKO, 10WT and 10AIDKO, 19WT and 19AIDKO by qRT-PCR. Data shown are representative of 3 technical replicates. (B) BCL6 protein levels were measured in 4WT and 4AIDKO, 10AIDKO and 19AIDKO cells by immunoblots, and GAPDH protein was taken as an internal control. (C) Schematic diagram shows AID mediated BCL6 mutation region. Not drawn to scale. (D) Histograms represent point mutation frequency of BCL6 promoter in 4WT and 4AIDKO cells. Mutation was measured in DNA from 2 independent preparations. (E) Schematic diagram shows CpG sites in the promoter of BCL6 gene. The hollow box represents BCL6 promoter. The shadow boxes represent CpG sites. The segment for 17 CpG sites locates in −1.07 kb to −0.84 kb of BCL6 promoter. The segment for 4 CpG sites is in the −0.51 kb to −0.24 kb of BCL6 promoter. Not drawn to scale. The bisulfite sequencing was used to examine methylation status of these CpG sites in BCL6 promoter. Black circles signify methylated CpGs, and white circles indicate demethylated CpGs. (F) Histograms indicate results for demethylation frequency of CpG sites in BCL6 promoter as detecting in E. Methylation was measured in DNA from 2 independent preparations. Data are presented as mean ± SD. *,** and *** represent p

    Techniques Used: Quantitative RT-PCR, Western Blot, Mutagenesis, Methylation Sequencing, Methylation

    AID-DNMT1 complex binds to BCL6 promoter. (A, B) Enrichments of AID binding (A) and DNMT1 binding (B) to the two sites of BCL6 promoter in 4WT and 4AIDKO, 10WT and 10AIDKO, 19WT and 19AIDKO cells, respectively. Chromatin Immunoprecipitation (ChIP) was performed using anti-AID and anti-DNMT1 following quantitative PCR amplifying two binding sites from ChIP enriched DNA. Data were collected from 3 independent experiments. Data shown are representative of 3 technical replicates. (C) Schematic diagram shows the indicated regions amplified from the BCL6 promoter to prepare the constructs for luciferase activity assay, the hollow boxes indicates the amplified fragments, shadow boxes represents pGL3-basic elements. p, a 1.8 kb BCL6 promoter (positions −1.8 kb to 0 kb); p1, p2, p3, p4 represents 0.4 kb (positions −0.4 kb to 0 kb), 0.5 kb (positions −0.9 kb to −0.4 kb), 0.5 kb (positions −1.4 kb to −0.9 kb), 0.4 kb (positions −1.8 kb to −1.4 kb) regions in BCL6 promoter. (D, E, F) Luciferase activity in 4WT and 4AIDKO (D), 10WT and 10AIDKO (E), 19WT and 19AIDKO (F) cells transfected with PGL3-basic vector including the indicated BC L6 promoter fragments was detected, the results were shown as histograms. Data shown are representative of 3 independent experiments. (G, H, I) Luciferase activity assay of PGL3-basic vector with the indicated BCL6 promoter fragments in 4WT and 4DNMT1KO (G), 10WT and 10DNMT1KO (H), 19WT and 19DNMT1KO (I) cells was performed, the results were shown as histograms. Data shown are representative of 3 independent experiments. Data are represented as mean ± SD. *,** and *** represent p
    Figure Legend Snippet: AID-DNMT1 complex binds to BCL6 promoter. (A, B) Enrichments of AID binding (A) and DNMT1 binding (B) to the two sites of BCL6 promoter in 4WT and 4AIDKO, 10WT and 10AIDKO, 19WT and 19AIDKO cells, respectively. Chromatin Immunoprecipitation (ChIP) was performed using anti-AID and anti-DNMT1 following quantitative PCR amplifying two binding sites from ChIP enriched DNA. Data were collected from 3 independent experiments. Data shown are representative of 3 technical replicates. (C) Schematic diagram shows the indicated regions amplified from the BCL6 promoter to prepare the constructs for luciferase activity assay, the hollow boxes indicates the amplified fragments, shadow boxes represents pGL3-basic elements. p, a 1.8 kb BCL6 promoter (positions −1.8 kb to 0 kb); p1, p2, p3, p4 represents 0.4 kb (positions −0.4 kb to 0 kb), 0.5 kb (positions −0.9 kb to −0.4 kb), 0.5 kb (positions −1.4 kb to −0.9 kb), 0.4 kb (positions −1.8 kb to −1.4 kb) regions in BCL6 promoter. (D, E, F) Luciferase activity in 4WT and 4AIDKO (D), 10WT and 10AIDKO (E), 19WT and 19AIDKO (F) cells transfected with PGL3-basic vector including the indicated BC L6 promoter fragments was detected, the results were shown as histograms. Data shown are representative of 3 independent experiments. (G, H, I) Luciferase activity assay of PGL3-basic vector with the indicated BCL6 promoter fragments in 4WT and 4DNMT1KO (G), 10WT and 10DNMT1KO (H), 19WT and 19DNMT1KO (I) cells was performed, the results were shown as histograms. Data shown are representative of 3 independent experiments. Data are represented as mean ± SD. *,** and *** represent p

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Construct, Luciferase, Activity Assay, Transfection, Plasmid Preparation

    40) Product Images from "Spermidine Synthase Genes Are Essential for Survival of Arabidopsis"

    Article Title: Spermidine Synthase Genes Are Essential for Survival of Arabidopsis

    Journal: Plant Physiology

    doi: 10.1104/pp.104.041699

    T-DNA insertion mutants of SPDS1 and SPDS2 . A, Schematic diagrams of the spds1-1 , spds2-1 , and spds2-2 alleles indicating locations of the T-DNA insertion. The positions and lengths of exons and introns are indicated by black rectangles and lines, respectively. B, RT-PCR analysis of SPDS1 expression in wild-type (WT) and spds1-1 plants. C, RT-PCR analysis of SPDS2 expression in wild-type, spds2-1 , and spds2-2 plants. Total RNA was prepared from 10-d-old seedlings. The level of Actin8 ( ACT8 ) was used as an internal control.
    Figure Legend Snippet: T-DNA insertion mutants of SPDS1 and SPDS2 . A, Schematic diagrams of the spds1-1 , spds2-1 , and spds2-2 alleles indicating locations of the T-DNA insertion. The positions and lengths of exons and introns are indicated by black rectangles and lines, respectively. B, RT-PCR analysis of SPDS1 expression in wild-type (WT) and spds1-1 plants. C, RT-PCR analysis of SPDS2 expression in wild-type, spds2-1 , and spds2-2 plants. Total RNA was prepared from 10-d-old seedlings. The level of Actin8 ( ACT8 ) was used as an internal control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Related Articles

    Amplification:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
    Article Snippet: .. RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. .. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file .

    Article Title: Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms
    Article Snippet: .. The 5’ flanking region of the sense transcripts of SlLNR1 were obtained by RNA ligase-mediated rapid amplification of 5' cDNA ends First Choice ® RLM-RACE Kit (Invitrogen, USA), according to the instructions of the manufacturer, and the 3’ end was verified by 3’ RACE PCR kit (TAKARA). .. Full sense transcript of the SlLNR1 was amplified with the primers designed according to the joint sequence by RT-PCR.

    Article Title: Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation
    Article Snippet: .. The sequence of the gene coding the gametocyte protein was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the RNA LA PCR Kit (TaKaRa Bio. ..

    Synthesized:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
    Article Snippet: .. RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. .. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file .

    Isolation:

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
    Article Snippet: .. RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. .. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file .

    Produced:

    Article Title: The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori
    Article Snippet: .. RT-PCR was performed using the LA RNA PCR kit (Takara, www.takara-bio.co.jp ) following the manufacturer's instructions. cDNA was produced by random priming. .. RT-PCR primers were as follows: endogenous BmA3 , BmA3QPCR1F (5′-TACAATGAGCTGCGTGTCG-3′) and BmA3QPCR1R (5′-CGGGCGTGTTGAATGTTTC -3′); and Bmdsx mRNA transcribed from the transgene, TGM2F (5′-ATTGGCGGGACACGATC-3′) and TGM2R (5′-AGCGCTCCGTAGCACAA-3′).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
    Article Snippet: .. RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. .. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file .

    Article Title: The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori
    Article Snippet: .. RT-PCR was performed using the LA RNA PCR kit (Takara, www.takara-bio.co.jp ) following the manufacturer's instructions. cDNA was produced by random priming. .. RT-PCR primers were as follows: endogenous BmA3 , BmA3QPCR1F (5′-TACAATGAGCTGCGTGTCG-3′) and BmA3QPCR1R (5′-CGGGCGTGTTGAATGTTTC -3′); and Bmdsx mRNA transcribed from the transgene, TGM2F (5′-ATTGGCGGGACACGATC-3′) and TGM2R (5′-AGCGCTCCGTAGCACAA-3′).

    Article Title: Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation
    Article Snippet: .. The sequence of the gene coding the gametocyte protein was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the RNA LA PCR Kit (TaKaRa Bio. ..

    Article Title: Spermidine Synthase Genes Are Essential for Survival of Arabidopsis
    Article Snippet: .. RT-PCR was conducted by using the RNA LA PCR Kit (Takara, Kyoto) with 0.5 μ g of total RNA. ..

    Random Hexamer Labeling:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Polymerase Chain Reaction:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
    Article Snippet: .. RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. .. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file .

    Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
    Article Snippet: .. Primer pairs specific for Tn4351 (primers TN-1 and IS4351-F) were used to amplify the sequences adjacent to the insertion site using the LA PCR kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). .. The nucleotide sequence was compared to sequence in the National Center for Biotechnology Information database using the BLASTX program [ ].

    Article Title: The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori
    Article Snippet: .. RT-PCR was performed using the LA RNA PCR kit (Takara, www.takara-bio.co.jp ) following the manufacturer's instructions. cDNA was produced by random priming. .. RT-PCR primers were as follows: endogenous BmA3 , BmA3QPCR1F (5′-TACAATGAGCTGCGTGTCG-3′) and BmA3QPCR1R (5′-CGGGCGTGTTGAATGTTTC -3′); and Bmdsx mRNA transcribed from the transgene, TGM2F (5′-ATTGGCGGGACACGATC-3′) and TGM2R (5′-AGCGCTCCGTAGCACAA-3′).

    Article Title: Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms
    Article Snippet: .. The 5’ flanking region of the sense transcripts of SlLNR1 were obtained by RNA ligase-mediated rapid amplification of 5' cDNA ends First Choice ® RLM-RACE Kit (Invitrogen, USA), according to the instructions of the manufacturer, and the 3’ end was verified by 3’ RACE PCR kit (TAKARA). .. Full sense transcript of the SlLNR1 was amplified with the primers designed according to the joint sequence by RT-PCR.

    Article Title: Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation
    Article Snippet: .. The sequence of the gene coding the gametocyte protein was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the RNA LA PCR Kit (TaKaRa Bio. ..

    Article Title: Spermidine Synthase Genes Are Essential for Survival of Arabidopsis
    Article Snippet: .. RT-PCR was conducted by using the RNA LA PCR Kit (Takara, Kyoto) with 0.5 μ g of total RNA. ..

    Article Title: Characterization of a novel germline PALB2 duplication in a hereditary breast and ovarian cancer family
    Article Snippet: .. LR-PCR was performed using a forward primer in intron 12 and a reverse primer in 3′-untranslated region (UTR) of PALB2 and the TaKaRa LA PCR kit (TaKaRa, Clontech) following the manufacturer’s instructions. ..

    Sequencing:

    Article Title: Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation
    Article Snippet: .. The sequence of the gene coding the gametocyte protein was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the RNA LA PCR Kit (TaKaRa Bio. ..

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  • 93
    TaKaRa long range lr pcr
    Clinical and molecular characterization of the case. (A) Frontal and lateral view of the proband at the age of 36 months. Note the mild frontal bossing, low-set posteriorly rotated ears, and thin lips. Earing aids are in place. The patient also displays brachydactyly of both hands and feet and clinodactily of V finger of hands. Other clinical findings are listed in the side table, where 3 out of 6 NH-CSS for SRS are in bold characters. (B) The <t>LR-PCR</t> amplicons of the patient (P) and both her father (F) and mother (M) were resolved on a 0.8% agarose gel. The patient (P) displayed a unique shorter band of about 2.5 kb in size, the father (F) showed a unique band of over 10 kb in size, corresponding to the expected 14.7 kb wild type band, whereas her mother (M) showed two bands corresponding to the wild type and the deleted alleles. MW, molecular weight. (C) The proximal and distal breakpoints of the SLC26A4 intragenic deletion were mapped within SLC26A4 IVS16 and the IVS20, respectively. The deletion is about 13 kb long. Sequence alignments of the junction fragments revealed an insertion of a part of CCDC126 IVS3. The rejoining between SLC26A4 IVS20 and CCDC126 IVS3 distal <t>bkp</t> occurred through a de novo 3 bp GCC insertion. (D) Sequencing of the RT-PCR amplicons, extending from exons 13–14 to 3′UTR of SLC26A4 , confirmed the homozygous deletion of exons 17–20 in the child (P). The father (F) showed only the long transcript corresponding to the wild type pendrin, whereas the mother (M) displayed a short transcript and a long one, consistent with a heterozygous state of the deletion. MW, molecular weight.
    Long Range Lr Pcr, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa la pcr kit
    Identification of the mutant strain RA1062. A <t>PCR</t> amplification. M: <t>Takara</t> DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p
    La Pcr Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa la dna polymerase
    ( A ) Gel electrophoresis of near full-length genome PCR products produced from four long amplifying <t>DNA</t> polymerases; KlenTaq LA (discontinued), AccuTaq LA, PrimeSTAR GXL and <t>Takara</t> LA Taq (separate gel) per manufacturer’s instructions for samples with high GC/secondary structures. M, HyperLadder 1 kb; 1, CVB3 Nancy; 2, CVB5 Faulkner; 3, H 2 O control. ( B ) Gel electrophoresis of near full-length genome PCR products produced from Takara LA Taq DNA polymerase. M, HyperLadder 1 kb; 1–4, known EV positives from NSW Health Pathology East virology diagnostic lab; 5, CVB3 Nancy; 6, H 2 .
    La Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Clinical and molecular characterization of the case. (A) Frontal and lateral view of the proband at the age of 36 months. Note the mild frontal bossing, low-set posteriorly rotated ears, and thin lips. Earing aids are in place. The patient also displays brachydactyly of both hands and feet and clinodactily of V finger of hands. Other clinical findings are listed in the side table, where 3 out of 6 NH-CSS for SRS are in bold characters. (B) The LR-PCR amplicons of the patient (P) and both her father (F) and mother (M) were resolved on a 0.8% agarose gel. The patient (P) displayed a unique shorter band of about 2.5 kb in size, the father (F) showed a unique band of over 10 kb in size, corresponding to the expected 14.7 kb wild type band, whereas her mother (M) showed two bands corresponding to the wild type and the deleted alleles. MW, molecular weight. (C) The proximal and distal breakpoints of the SLC26A4 intragenic deletion were mapped within SLC26A4 IVS16 and the IVS20, respectively. The deletion is about 13 kb long. Sequence alignments of the junction fragments revealed an insertion of a part of CCDC126 IVS3. The rejoining between SLC26A4 IVS20 and CCDC126 IVS3 distal bkp occurred through a de novo 3 bp GCC insertion. (D) Sequencing of the RT-PCR amplicons, extending from exons 13–14 to 3′UTR of SLC26A4 , confirmed the homozygous deletion of exons 17–20 in the child (P). The father (F) showed only the long transcript corresponding to the wild type pendrin, whereas the mother (M) displayed a short transcript and a long one, consistent with a heterozygous state of the deletion. MW, molecular weight.

    Journal: Frontiers in Genetics

    Article Title: Segmental Maternal UPD of Chromosome 7q in a Patient With Pendred and Silver Russell Syndromes-Like Features

    doi: 10.3389/fgene.2018.00600

    Figure Lengend Snippet: Clinical and molecular characterization of the case. (A) Frontal and lateral view of the proband at the age of 36 months. Note the mild frontal bossing, low-set posteriorly rotated ears, and thin lips. Earing aids are in place. The patient also displays brachydactyly of both hands and feet and clinodactily of V finger of hands. Other clinical findings are listed in the side table, where 3 out of 6 NH-CSS for SRS are in bold characters. (B) The LR-PCR amplicons of the patient (P) and both her father (F) and mother (M) were resolved on a 0.8% agarose gel. The patient (P) displayed a unique shorter band of about 2.5 kb in size, the father (F) showed a unique band of over 10 kb in size, corresponding to the expected 14.7 kb wild type band, whereas her mother (M) showed two bands corresponding to the wild type and the deleted alleles. MW, molecular weight. (C) The proximal and distal breakpoints of the SLC26A4 intragenic deletion were mapped within SLC26A4 IVS16 and the IVS20, respectively. The deletion is about 13 kb long. Sequence alignments of the junction fragments revealed an insertion of a part of CCDC126 IVS3. The rejoining between SLC26A4 IVS20 and CCDC126 IVS3 distal bkp occurred through a de novo 3 bp GCC insertion. (D) Sequencing of the RT-PCR amplicons, extending from exons 13–14 to 3′UTR of SLC26A4 , confirmed the homozygous deletion of exons 17–20 in the child (P). The father (F) showed only the long transcript corresponding to the wild type pendrin, whereas the mother (M) displayed a short transcript and a long one, consistent with a heterozygous state of the deletion. MW, molecular weight.

    Article Snippet: To confirm the deletion and localize its breakpoints (bkp) at nucleotide level, long-range (LR) PCR spanning the SLC26A4 genomic region from exon 16 to exon 21 was carried out with Takara LA Taq (Diatech, Jesi, Italy), according to cycle conditions suggested by the manufacturer.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Sequencing, Reverse Transcription Polymerase Chain Reaction

    Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p

    Journal: Veterinary Research

    Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

    doi: 10.1186/s13567-018-0589-8

    Figure Lengend Snippet: Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p

    Article Snippet: Primer pairs specific for Tn4351 (primers TN-1 and IS4351-F) were used to amplify the sequences adjacent to the insertion site using the LA PCR kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Marker, Southern Blot, Positive Control, Negative Control, Agarose Gel Electrophoresis, Labeling, Real-time Polymerase Chain Reaction, Expressing

    Pre-mRNA transcribed from the Bmdsx mini gene is sex-specifically spliced. (A) The diagram shows the structure of the Bmdsx minigene. Open boxes: common exons. Shaded boxes: female-specific exons. Arrows: primers for PCR. (B) Poly (A)+RNA was extracted from the fat body of transgenic silkworms. These RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers TGM2F and TGM2R. Resulting products were separated on a 1% agarose gel and visualized with SYBR Green I (Molecular Probes) at a dilution of 1: 10,000. M represents the DNA marker(λ /Hind III+ φX174/ Hinc II). The bands for the PCR products are schematically shown. (C) Poly (A)+RNA was extracted from the fat body. Upper lanes: these RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers TGM2F and TGM2R. Lower lanes: these RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers BmA3QPCR1F and BmA3QPCR1R. Resulting products were separated on a 1% agarose gel and visualized with SYBR Green I (Molecular Probes) at a dilution of 1: 10,000 (upper lanes) and visualized with ethidium bromide (lower lanes). M represents the DNA marker (λ /Hind III+ φX174/ Hinc II). The bands for the PCR products are schematically shown.

    Journal: Journal of Insect Science

    Article Title: The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori

    doi:

    Figure Lengend Snippet: Pre-mRNA transcribed from the Bmdsx mini gene is sex-specifically spliced. (A) The diagram shows the structure of the Bmdsx minigene. Open boxes: common exons. Shaded boxes: female-specific exons. Arrows: primers for PCR. (B) Poly (A)+RNA was extracted from the fat body of transgenic silkworms. These RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers TGM2F and TGM2R. Resulting products were separated on a 1% agarose gel and visualized with SYBR Green I (Molecular Probes) at a dilution of 1: 10,000. M represents the DNA marker(λ /Hind III+ φX174/ Hinc II). The bands for the PCR products are schematically shown. (C) Poly (A)+RNA was extracted from the fat body. Upper lanes: these RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers TGM2F and TGM2R. Lower lanes: these RNAs were reverse-transcribed with random hexamer, and the cDNAs were PCR-amplified with primers BmA3QPCR1F and BmA3QPCR1R. Resulting products were separated on a 1% agarose gel and visualized with SYBR Green I (Molecular Probes) at a dilution of 1: 10,000 (upper lanes) and visualized with ethidium bromide (lower lanes). M represents the DNA marker (λ /Hind III+ φX174/ Hinc II). The bands for the PCR products are schematically shown.

    Article Snippet: RT-PCR was performed using the LA RNA PCR kit (Takara, www.takara-bio.co.jp ) following the manufacturer's instructions. cDNA was produced by random priming.

    Techniques: Polymerase Chain Reaction, Transgenic Assay, Random Hexamer Labeling, Amplification, Agarose Gel Electrophoresis, SYBR Green Assay, Marker

    ( A ) Gel electrophoresis of near full-length genome PCR products produced from four long amplifying DNA polymerases; KlenTaq LA (discontinued), AccuTaq LA, PrimeSTAR GXL and Takara LA Taq (separate gel) per manufacturer’s instructions for samples with high GC/secondary structures. M, HyperLadder 1 kb; 1, CVB3 Nancy; 2, CVB5 Faulkner; 3, H 2 O control. ( B ) Gel electrophoresis of near full-length genome PCR products produced from Takara LA Taq DNA polymerase. M, HyperLadder 1 kb; 1–4, known EV positives from NSW Health Pathology East virology diagnostic lab; 5, CVB3 Nancy; 6, H 2 .

    Journal: Scientific Reports

    Article Title: Amplification and next generation sequencing of near full-length human enteroviruses for identification and characterisation from clinical samples

    doi: 10.1038/s41598-018-30322-y

    Figure Lengend Snippet: ( A ) Gel electrophoresis of near full-length genome PCR products produced from four long amplifying DNA polymerases; KlenTaq LA (discontinued), AccuTaq LA, PrimeSTAR GXL and Takara LA Taq (separate gel) per manufacturer’s instructions for samples with high GC/secondary structures. M, HyperLadder 1 kb; 1, CVB3 Nancy; 2, CVB5 Faulkner; 3, H 2 O control. ( B ) Gel electrophoresis of near full-length genome PCR products produced from Takara LA Taq DNA polymerase. M, HyperLadder 1 kb; 1–4, known EV positives from NSW Health Pathology East virology diagnostic lab; 5, CVB3 Nancy; 6, H 2 .

    Article Snippet: First round PCR was performed by adding cDNA (5 µL) to a reaction mix (50 µL) containing Takara LA Buffer (1X), dNTPs (100 µM each), forward primer vir24 (0.2 µM), reverse primer vir20 (0.2 µM), nuclease-free water (29.5 µL) and Takara LA DNA polymerase (2.5 U).

    Techniques: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Produced, Diagnostic Assay