spei  (New England Biolabs)


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    Structured Review

    New England Biolabs spei
    OSN-specific GABAR B1 and D2R knockout mice, Related to Figure 2 . (A) Gene targeting at Drd2 locus. We obtained germline transmission from one ES clone, HEPD064_5_D11. The Drd2 locus in Drd2 tm1a , Drd2 fl (= Drd tm1c ) and Drd cKO . Drd2 +/ tm1a mice were crossed with Flp mice to obtain Drd2 +/ fl . ( C ) Gene targeting at Gabbr1 locus. We obtained germline transmission from one ES clone, EPD0730_1_G04. ( D ) The Gabbr1 locus in Gabbr1 tm1a , Gabbr1 fl (= Gabbr1 tm1c ), and Gabbr1 cKO . Gabbr1 +/ tm1a mice were crossed with Flp mice to obtain Gabbr1 +/ fl . ( E-G ) Southern blot analysis on Drd2 +/+ and Drd2 tm1a /+ ( E , Drd2 probe), Gabbr1 +/+ and Gabbr1 tm1a /+ ( F , Gabbr1 probe); Drd2 tm1a /+ and Gabbr1 tm1a /+ ( G , neo r probe). ( H ) Immunostaining of D2R in the OB of OSN-specific Drd2 mutant mice. ( I ) Immunostaining of GABAR B1 in the OB of OSN-specific Gabbr1 mutant mice. Location of 5’ and 3’ arms for gene targeting and 5’ and 3’ DNA probes for southern blotting are shown in ( A ) and ( C ). <t>KpnI</t> (K), <t>SpeI</t> (S), XhoI (X) and ApaI (A).
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    Images

    1) Product Images from "Widespread inhibition, antagonism, and synergy in mouse olfactory sensory neurons in vivo"

    Article Title: Widespread inhibition, antagonism, and synergy in mouse olfactory sensory neurons in vivo

    Journal: bioRxiv

    doi: 10.1101/803908

    OSN-specific GABAR B1 and D2R knockout mice, Related to Figure 2 . (A) Gene targeting at Drd2 locus. We obtained germline transmission from one ES clone, HEPD064_5_D11. The Drd2 locus in Drd2 tm1a , Drd2 fl (= Drd tm1c ) and Drd cKO . Drd2 +/ tm1a mice were crossed with Flp mice to obtain Drd2 +/ fl . ( C ) Gene targeting at Gabbr1 locus. We obtained germline transmission from one ES clone, EPD0730_1_G04. ( D ) The Gabbr1 locus in Gabbr1 tm1a , Gabbr1 fl (= Gabbr1 tm1c ), and Gabbr1 cKO . Gabbr1 +/ tm1a mice were crossed with Flp mice to obtain Gabbr1 +/ fl . ( E-G ) Southern blot analysis on Drd2 +/+ and Drd2 tm1a /+ ( E , Drd2 probe), Gabbr1 +/+ and Gabbr1 tm1a /+ ( F , Gabbr1 probe); Drd2 tm1a /+ and Gabbr1 tm1a /+ ( G , neo r probe). ( H ) Immunostaining of D2R in the OB of OSN-specific Drd2 mutant mice. ( I ) Immunostaining of GABAR B1 in the OB of OSN-specific Gabbr1 mutant mice. Location of 5’ and 3’ arms for gene targeting and 5’ and 3’ DNA probes for southern blotting are shown in ( A ) and ( C ). KpnI (K), SpeI (S), XhoI (X) and ApaI (A).
    Figure Legend Snippet: OSN-specific GABAR B1 and D2R knockout mice, Related to Figure 2 . (A) Gene targeting at Drd2 locus. We obtained germline transmission from one ES clone, HEPD064_5_D11. The Drd2 locus in Drd2 tm1a , Drd2 fl (= Drd tm1c ) and Drd cKO . Drd2 +/ tm1a mice were crossed with Flp mice to obtain Drd2 +/ fl . ( C ) Gene targeting at Gabbr1 locus. We obtained germline transmission from one ES clone, EPD0730_1_G04. ( D ) The Gabbr1 locus in Gabbr1 tm1a , Gabbr1 fl (= Gabbr1 tm1c ), and Gabbr1 cKO . Gabbr1 +/ tm1a mice were crossed with Flp mice to obtain Gabbr1 +/ fl . ( E-G ) Southern blot analysis on Drd2 +/+ and Drd2 tm1a /+ ( E , Drd2 probe), Gabbr1 +/+ and Gabbr1 tm1a /+ ( F , Gabbr1 probe); Drd2 tm1a /+ and Gabbr1 tm1a /+ ( G , neo r probe). ( H ) Immunostaining of D2R in the OB of OSN-specific Drd2 mutant mice. ( I ) Immunostaining of GABAR B1 in the OB of OSN-specific Gabbr1 mutant mice. Location of 5’ and 3’ arms for gene targeting and 5’ and 3’ DNA probes for southern blotting are shown in ( A ) and ( C ). KpnI (K), SpeI (S), XhoI (X) and ApaI (A).

    Techniques Used: Knock-Out, Mouse Assay, Transmission Assay, Southern Blot, Immunostaining, Mutagenesis

    2) Product Images from "Soluble L1CAM promotes breast cancer cell adhesion and migration in vitro, but not invasion"

    Article Title: Soluble L1CAM promotes breast cancer cell adhesion and migration in vitro, but not invasion

    Journal: Cancer Cell International

    doi: 10.1186/1475-2867-10-34

    Over-expressing L1-ectodomain in MDA-MB-468 cells . (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via SpeI and XhoI restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).
    Figure Legend Snippet: Over-expressing L1-ectodomain in MDA-MB-468 cells . (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via SpeI and XhoI restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).

    Techniques Used: Expressing, Multiple Displacement Amplification, Plasmid Preparation, Amplification, Construct, Stable Transfection, Immunostaining, FACS, Infection, TCA Precipitation, Western Blot

    3) Product Images from "RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation"

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp780

    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Figure Legend Snippet: Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.

    Techniques Used: Southern Blot, Staining, Produced, Generated

    4) Product Images from "Production of enterodiol from defatted flaxseeds through biotransformation by human intestinal bacteria"

    Article Title: Production of enterodiol from defatted flaxseeds through biotransformation by human intestinal bacteria

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-10-115

    PFGE patterns of SpeI-cleaved genomic DNA of 32 pure cultures obtained from END-49 . Assignment of the bacterial strains to Genome Group I, II, III, IV or V was indicated at the bottom of the PFGE photo.
    Figure Legend Snippet: PFGE patterns of SpeI-cleaved genomic DNA of 32 pure cultures obtained from END-49 . Assignment of the bacterial strains to Genome Group I, II, III, IV or V was indicated at the bottom of the PFGE photo.

    Techniques Used:

    5) Product Images from "Transmission of Yersinia pseudotuberculosis in the Pork Production Chain from Farm to Slaughterhouse ▿"

    Article Title: Transmission of Yersinia pseudotuberculosis in the Pork Production Chain from Farm to Slaughterhouse ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02664-07

    Seven different SpeI profiles (lanes 1 to 7) of Yersinia pseudotuberculosis strains obtained. M designates midrange PFGE markers.
    Figure Legend Snippet: Seven different SpeI profiles (lanes 1 to 7) of Yersinia pseudotuberculosis strains obtained. M designates midrange PFGE markers.

    Techniques Used:

    6) Product Images from "Simultaneous Detection and Mutation Surveillance of SARS-CoV-2 and co-infections of multiple respiratory viruses by Rapid field-deployable sequencing"

    Article Title: Simultaneous Detection and Mutation Surveillance of SARS-CoV-2 and co-infections of multiple respiratory viruses by Rapid field-deployable sequencing

    Journal: medRxiv

    doi: 10.1101/2020.06.12.20129247

    Agarose gel electrophoresis results of singleplex RPA a , Agarose gel electrophoresis results of singleplex RPA with selected primers shown next a molecular size marker. The amplicons range from 194 bp to 466 bp. b , Agarose gel electrophoresis results of restriction enzyme digestion. The amplicon of pair 5 was digested by SpeI while the others were digested by NlaIII. The digested DNA bands (asterisks) were of expected sizes. c , Agarose gel electrophoresis results showing the sensitivity of RPA in amplifying the SARS-CoV-2 genome. Primer pair 4 was used in the experiment. Reliable amplification can be achieved with 1.4 copies (calculated from dilution) of the SARS-CoV-2 genome. d , Agarose gel electrophoresis result of one-pot reverse transcription and RPA reaction using primer pair 4.
    Figure Legend Snippet: Agarose gel electrophoresis results of singleplex RPA a , Agarose gel electrophoresis results of singleplex RPA with selected primers shown next a molecular size marker. The amplicons range from 194 bp to 466 bp. b , Agarose gel electrophoresis results of restriction enzyme digestion. The amplicon of pair 5 was digested by SpeI while the others were digested by NlaIII. The digested DNA bands (asterisks) were of expected sizes. c , Agarose gel electrophoresis results showing the sensitivity of RPA in amplifying the SARS-CoV-2 genome. Primer pair 4 was used in the experiment. Reliable amplification can be achieved with 1.4 copies (calculated from dilution) of the SARS-CoV-2 genome. d , Agarose gel electrophoresis result of one-pot reverse transcription and RPA reaction using primer pair 4.

    Techniques Used: Agarose Gel Electrophoresis, Recombinase Polymerase Amplification, Marker, Amplification

    7) Product Images from "Seven novel mutations in the long isoform of the USH2A gene in Chinese families with nonsyndromic retinitis pigmentosa and Usher syndrome Type II"

    Article Title: Seven novel mutations in the long isoform of the USH2A gene in Chinese families with nonsyndromic retinitis pigmentosa and Usher syndrome Type II

    Journal: Molecular Vision

    doi:

    A restriction fragment length analysis of the four mutations detected in this study. A : c.2802T > G abolished a HincII restriction site that co-segregated with the affected individuals and the carriers (42 bp, 57 bp, 99 bp, 717 bp, and 774 bp), but not with unaffected individuals and normal controls (42 bp, 57 bp, and 717 bp). B : c.8232G > C created a new HpyCH4V restriction site that co-segregated with the affected individuals and the carriers (88 bp, 186 bp, 218 bp, and 274 bp), but not with unaffected individuals and normal controls (218 bp, 274 bp). C : c.3788G > A abolished a BsaI restriction site that co-segregated with the affected individuals and the carriers (70 bp, 132 bp, 422 bp, and 492 bp), but not with unaffected individuals and normal controls (70 bp, 132 bp, and 422 bp). D : c.14403C > G created a SpeI restriction site that co-segregated with the affected individuals and the carriers (145 bp, 300 bp, and 445 bp), but not with unaffected individuals and normal controls (445 bp). A participant identification number is given above each lane. N represents normal controls.
    Figure Legend Snippet: A restriction fragment length analysis of the four mutations detected in this study. A : c.2802T > G abolished a HincII restriction site that co-segregated with the affected individuals and the carriers (42 bp, 57 bp, 99 bp, 717 bp, and 774 bp), but not with unaffected individuals and normal controls (42 bp, 57 bp, and 717 bp). B : c.8232G > C created a new HpyCH4V restriction site that co-segregated with the affected individuals and the carriers (88 bp, 186 bp, 218 bp, and 274 bp), but not with unaffected individuals and normal controls (218 bp, 274 bp). C : c.3788G > A abolished a BsaI restriction site that co-segregated with the affected individuals and the carriers (70 bp, 132 bp, 422 bp, and 492 bp), but not with unaffected individuals and normal controls (70 bp, 132 bp, and 422 bp). D : c.14403C > G created a SpeI restriction site that co-segregated with the affected individuals and the carriers (145 bp, 300 bp, and 445 bp), but not with unaffected individuals and normal controls (445 bp). A participant identification number is given above each lane. N represents normal controls.

    Techniques Used:

    8) Product Images from "Molecular Epidemiologic Analysis and Antimicrobial Resistance of Helicobacter cinaedi Isolated from Seven Hospitals in Japan"

    Article Title: Molecular Epidemiologic Analysis and Antimicrobial Resistance of Helicobacter cinaedi Isolated from Seven Hospitals in Japan

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.06810-11

    Pulsed-field gel electrophoresis patterns of XhoI-digested (A) and SpeI-digested (B) H. cinaedi isolates from Japan as well as the six reference strains. The letter in each isolate's identifier indicates the hospital at which the strain was isolated (e.g.,
    Figure Legend Snippet: Pulsed-field gel electrophoresis patterns of XhoI-digested (A) and SpeI-digested (B) H. cinaedi isolates from Japan as well as the six reference strains. The letter in each isolate's identifier indicates the hospital at which the strain was isolated (e.g.,

    Techniques Used: Pulsed-Field Gel, Electrophoresis, Isolation

    9) Product Images from "Methylase-assisted subcloning for high throughput BioBrick assembly"

    Article Title: Methylase-assisted subcloning for high throughput BioBrick assembly

    Journal: PeerJ

    doi: 10.7717/peerj.9841

    Model plasmids used in this study. The lacI-Ptac-lacO insert (A) includes a promoter that is somewhat leaky at high copy number. The IMBB2.4-pUC57-mini backbone (A–B), hereafter abbreviated pUC, is BioBrick-compatible and also includes an NsiI site downstream of PstI ( Matsumura, 2017 ). The tagRFP reporter (B) protein can cause colonies to turn visibly pink, but only when the gene encoding it is subcloned downstream of a leaky or constitutive promoter. RP4 oriT-pUC-cat (C) is a BioBrick compatible plasmid that confers resistance to chloramphenicol instead of ampicillin. RP4 oriT serves as a small stuffer in these experiments. In this study this latter plasmid is used only as a recipient plasmid (destination vector) for 3A assembly. Five expression vectors for production of recombinant DNA methyltransferases were constructed for this study. The version that expresses M.Ocy1ORF8430P, a putative ortholog of M.SpeI, is shown (D). The others are similar in design but express M.XbaI, M.EcoRI, M.PstI or M.AvaIII instead. Each plasmid utilizes the low copy number p15A origin (pACYC) and confers resistance to spectinomycin and is thus compatible with pUC plasmids that impart resistance to ampicillin, chloramphenicol or kanamycin. The DNA methyltransferase expression vectors do not contain any of the restriction sites employed in BioBrick assembly protocols (EcoRI, XbaI, SpeI or PstI), so they will not produce restriction fragments that ligate to those that are desired.
    Figure Legend Snippet: Model plasmids used in this study. The lacI-Ptac-lacO insert (A) includes a promoter that is somewhat leaky at high copy number. The IMBB2.4-pUC57-mini backbone (A–B), hereafter abbreviated pUC, is BioBrick-compatible and also includes an NsiI site downstream of PstI ( Matsumura, 2017 ). The tagRFP reporter (B) protein can cause colonies to turn visibly pink, but only when the gene encoding it is subcloned downstream of a leaky or constitutive promoter. RP4 oriT-pUC-cat (C) is a BioBrick compatible plasmid that confers resistance to chloramphenicol instead of ampicillin. RP4 oriT serves as a small stuffer in these experiments. In this study this latter plasmid is used only as a recipient plasmid (destination vector) for 3A assembly. Five expression vectors for production of recombinant DNA methyltransferases were constructed for this study. The version that expresses M.Ocy1ORF8430P, a putative ortholog of M.SpeI, is shown (D). The others are similar in design but express M.XbaI, M.EcoRI, M.PstI or M.AvaIII instead. Each plasmid utilizes the low copy number p15A origin (pACYC) and confers resistance to spectinomycin and is thus compatible with pUC plasmids that impart resistance to ampicillin, chloramphenicol or kanamycin. The DNA methyltransferase expression vectors do not contain any of the restriction sites employed in BioBrick assembly protocols (EcoRI, XbaI, SpeI or PstI), so they will not produce restriction fragments that ligate to those that are desired.

    Techniques Used: Plasmid Preparation, Expressing, Recombinant, Construct, Low Copy Number

    Conventional subcloning of BioBrick-compatible parts. Recipient (1) and donor (2) plasmids both contain inserts bound by the same restriction sites (E = EcoRI, X = XbaI, S = SpeI, P = PstI). The recipient plasmid (1) is cut with SpeI and PstI, releasing a short stuffer fragment (or “snippet,” 3); the donor (2) is separately cut with XbaI and PstI, so that insert (5) is released from plasmid fragment (6). The fragments from both digests (3-6) are separated by agarose gel electrophoresis. The desired recipient fragment (4) and insert (5) are excised from the gel and subsequently purified; the unwanted stuffer (3) and donor plasmid fragment (6) are left in the gels, which are thrown away. The recipient fragment (4) and insert (5) are ligated together forming three products: the recipient plasmid homodimer (7), the insert homodimer (8) and desired insert-recipient plasmid heterodimer (9). Large inverted repeats (7-8) cannot replicate stably in vivo so the desired construct (9) is the only product capable of conferring antibiotic selection if the digests and ligations were efficient.
    Figure Legend Snippet: Conventional subcloning of BioBrick-compatible parts. Recipient (1) and donor (2) plasmids both contain inserts bound by the same restriction sites (E = EcoRI, X = XbaI, S = SpeI, P = PstI). The recipient plasmid (1) is cut with SpeI and PstI, releasing a short stuffer fragment (or “snippet,” 3); the donor (2) is separately cut with XbaI and PstI, so that insert (5) is released from plasmid fragment (6). The fragments from both digests (3-6) are separated by agarose gel electrophoresis. The desired recipient fragment (4) and insert (5) are excised from the gel and subsequently purified; the unwanted stuffer (3) and donor plasmid fragment (6) are left in the gels, which are thrown away. The recipient fragment (4) and insert (5) are ligated together forming three products: the recipient plasmid homodimer (7), the insert homodimer (8) and desired insert-recipient plasmid heterodimer (9). Large inverted repeats (7-8) cannot replicate stably in vivo so the desired construct (9) is the only product capable of conferring antibiotic selection if the digests and ligations were efficient.

    Techniques Used: Subcloning, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Stable Transfection, In Vivo, Construct, Selection

    4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .
    Figure Legend Snippet: 4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .

    Techniques Used: Plasmid Preparation, Methylation, In Vivo, Ligation, Recombinant

    10) Product Images from "Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H-/Hnt Isolates from Cattle and Clonal Relationship Analysis ▿ Serotype O156:H25/H-/Hnt Isolates from Cattle and Clonal Relationship Analysis ▿ †"

    Article Title: Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H-/Hnt Isolates from Cattle and Clonal Relationship Analysis ▿ Serotype O156:H25/H-/Hnt Isolates from Cattle and Clonal Relationship Analysis ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00743-10

    Neighbor-joining tree of bovine E. coli O156 isolates based on the restriction pattern obtained after digestion with XbaI, NotI, BlnI, and SpeI (see Fig. S1 in the supplemental material).
    Figure Legend Snippet: Neighbor-joining tree of bovine E. coli O156 isolates based on the restriction pattern obtained after digestion with XbaI, NotI, BlnI, and SpeI (see Fig. S1 in the supplemental material).

    Techniques Used:

    11) Product Images from "Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes"

    Article Title: Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-59745-483-4_40

    Converting ZFPs into ZFNs. The NdeI/SpeI-cut ZFPs are ligated into the pET15b:N, the plasmid containing the FokI cleavage domain to form pET15b:ZFN.
    Figure Legend Snippet: Converting ZFPs into ZFNs. The NdeI/SpeI-cut ZFPs are ligated into the pET15b:N, the plasmid containing the FokI cleavage domain to form pET15b:ZFN.

    Techniques Used: Plasmid Preparation

    12) Product Images from "Association of iss and iucA, but Not tsh, with Plasmid-Mediated Virulence of Avian Pathogenic Escherichia coli"

    Article Title: Association of iss and iucA, but Not tsh, with Plasmid-Mediated Virulence of Avian Pathogenic Escherichia coli

    Journal:

    doi: 10.1128/IAI.72.11.6554-6560.2004

    Physical map of plasmid pVM01::Tn phoA illustrating the NotI, SpeI, and XbaI restriction endonuclease cleavage sites and the locations of the Tn phoA insert and the putative virulence genes, iucA , iss , and tsh. Designation of fragments (A,B,C, or D) corresponds
    Figure Legend Snippet: Physical map of plasmid pVM01::Tn phoA illustrating the NotI, SpeI, and XbaI restriction endonuclease cleavage sites and the locations of the Tn phoA insert and the putative virulence genes, iucA , iss , and tsh. Designation of fragments (A,B,C, or D) corresponds

    Techniques Used: Plasmid Preparation

    13) Product Images from "Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae"

    Article Title: Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    Journal: Bioengineered

    doi: 10.4161/bioe.29167

    Figure 4. In vivo assembly of synthetic genes by IVOE. Each overlapping region allowed crossover events to occur between fragments giving rise to an autonomously repaired vector containing single and double expression cassettes in the correct orientation. ( A ) and ( B ) Single expression cassettes are constructed in pESC by engineering specific primers containing overhangs to foster in vivo cloning with the linearized plasmid in yeast (pJRoC30 was used as template for Vp or Lac amplifications). ( C ) and ( D ) The pESC constructs obtained in ( A ) and ( B ) were used as scaffolds to assemble Lac and Vp genes under the control of different promoter/terminator pairs. Primers used: (1)-MCS1- Vp / Lac -α-BamHI, (2)-MCS2- Vp -ter-NheI, (3)-MCS2- Lac -ter-NheI, (4)-MCS1- Vp / Lac -α-SpeI, (5)-MCS1- Lac -ter-SacI and (6)-MCS1- Vp -ter-SacI. Black arrows indicate the direction of the transcription process.
    Figure Legend Snippet: Figure 4. In vivo assembly of synthetic genes by IVOE. Each overlapping region allowed crossover events to occur between fragments giving rise to an autonomously repaired vector containing single and double expression cassettes in the correct orientation. ( A ) and ( B ) Single expression cassettes are constructed in pESC by engineering specific primers containing overhangs to foster in vivo cloning with the linearized plasmid in yeast (pJRoC30 was used as template for Vp or Lac amplifications). ( C ) and ( D ) The pESC constructs obtained in ( A ) and ( B ) were used as scaffolds to assemble Lac and Vp genes under the control of different promoter/terminator pairs. Primers used: (1)-MCS1- Vp / Lac -α-BamHI, (2)-MCS2- Vp -ter-NheI, (3)-MCS2- Lac -ter-NheI, (4)-MCS1- Vp / Lac -α-SpeI, (5)-MCS1- Lac -ter-SacI and (6)-MCS1- Vp -ter-SacI. Black arrows indicate the direction of the transcription process.

    Techniques Used: In Vivo, Plasmid Preparation, Expressing, Construct, Clone Assay

    14) Product Images from "Replacement of Glycoprotein B in Alcelaphine Herpesvirus 1 by Its Ovine Herpesvirus 2 Homolog : Implications in Vaccine Development for Sheep-Associated Malignant Catarrhal Fever"

    Article Title: Replacement of Glycoprotein B in Alcelaphine Herpesvirus 1 by Its Ovine Herpesvirus 2 Homolog : Implications in Vaccine Development for Sheep-Associated Malignant Catarrhal Fever

    Journal: mSphere

    doi: 10.1128/mSphere.00108-16

    Replacement of AlHV-1 ORF8 by OvHV-2 ORF8 in the AlHV-1 ΔORF73 BAC using the galK recombineering system. (Top) In step 1, the galK gene sequence flanked by arms (R1 and R2) corresponding to AlHV-1 ORF8 was produced by PCR and transformed into E. coli SW102 containing the AlHV-1 ΔORF73 BAC. Positive selection on minimal medium containing galactose was used to identify colonies carrying the galK gene (AlHV-1 ΔORF73 ΔORF8 ). In step 2, a clone with the AlHV-1 ORF8 replaced by galK was subjected to recombination with a PCR fragment containing the OvHV-2 ORF8 gene flanked by the R1 and R2 arms. Negative selection was performed on plates containing minimal medium and 2-deoxygalactose with glycerol as the carbon source and clones carrying OvHV-2 ORF8 (AlHV-1 ΔORF73/OvHV-2-ORF8 ) were selected. (Bottom) Restriction patterns of the BAC DNAs are demonstrated following digestion with SpeI and EcoRI and electrophoresis in 1% agarose/SYBR green gel. Lanes: 1, AlHV-1 ΔORF73 ; 2, AlHV-1 ΔORF73 ΔORF8 ; 3, AlHV-1 ΔORF73/OvHV-2-ORF8 . Red arrows indicate shifts in band size or new bands. The expected sizes of target bands are indicated in base pairs to the right of the gels.
    Figure Legend Snippet: Replacement of AlHV-1 ORF8 by OvHV-2 ORF8 in the AlHV-1 ΔORF73 BAC using the galK recombineering system. (Top) In step 1, the galK gene sequence flanked by arms (R1 and R2) corresponding to AlHV-1 ORF8 was produced by PCR and transformed into E. coli SW102 containing the AlHV-1 ΔORF73 BAC. Positive selection on minimal medium containing galactose was used to identify colonies carrying the galK gene (AlHV-1 ΔORF73 ΔORF8 ). In step 2, a clone with the AlHV-1 ORF8 replaced by galK was subjected to recombination with a PCR fragment containing the OvHV-2 ORF8 gene flanked by the R1 and R2 arms. Negative selection was performed on plates containing minimal medium and 2-deoxygalactose with glycerol as the carbon source and clones carrying OvHV-2 ORF8 (AlHV-1 ΔORF73/OvHV-2-ORF8 ) were selected. (Bottom) Restriction patterns of the BAC DNAs are demonstrated following digestion with SpeI and EcoRI and electrophoresis in 1% agarose/SYBR green gel. Lanes: 1, AlHV-1 ΔORF73 ; 2, AlHV-1 ΔORF73 ΔORF8 ; 3, AlHV-1 ΔORF73/OvHV-2-ORF8 . Red arrows indicate shifts in band size or new bands. The expected sizes of target bands are indicated in base pairs to the right of the gels.

    Techniques Used: BAC Assay, Sequencing, Produced, Polymerase Chain Reaction, Transformation Assay, Selection, Clone Assay, Electrophoresis, SYBR Green Assay

    15) Product Images from "CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci"

    Article Title: CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci

    Journal: bioRxiv

    doi: 10.1101/162156

    CRISPR/Cas9-mediated knock-in of an optimized TetO repeat using linear donor DNA with short homology arms. (A) Schematic of the strategy to create linear donor DNA with short homology arms via PCR. The final plasmid used as a template is shown together with the primers used for amplification of the linear donor DNA (black arrows). Blasticidin resistance ( BlaR ) gene under EFS promoter was used for positive selection. The selection cassette is flanked with loxP sites in case it needs to be removed. (B) Schematic of the complete initial cassette containing three TetO repeats (green), four random 10 bp (light blue) sequences, three restriction sites StyI, SpeI, and BamHI (all of them are in red), and primers (dark blue) used for synthesis of the complementary strand and amplification of the product. (C) Schematic of the insertion of 3mer TetO cassette into pSP2 vector through StyI and BamHI sites. Each TetO sequence is indicated with a green box. (D) Schematic of the assembly of the TetO repeat multimers. StyI/SpeI site, created by insertion of the TetO cassette, cannot be cut by either of these two enzymes. (E) Results of the PCRs to amplify 48-mer and 96-mer TetO repeat donors for the HSP70 locus. The expected band sizes were around 3 kb for the 48-mer and around 4.6 kb for the 96-mer TetO donor. NEB 1 kb ladder (M) was used. (F) Schematic of the knock-in strategy. Homology arms (HA) are shown in purple. sgRNA target site is indicated with a red triangle. DSB: double strand break.
    Figure Legend Snippet: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat using linear donor DNA with short homology arms. (A) Schematic of the strategy to create linear donor DNA with short homology arms via PCR. The final plasmid used as a template is shown together with the primers used for amplification of the linear donor DNA (black arrows). Blasticidin resistance ( BlaR ) gene under EFS promoter was used for positive selection. The selection cassette is flanked with loxP sites in case it needs to be removed. (B) Schematic of the complete initial cassette containing three TetO repeats (green), four random 10 bp (light blue) sequences, three restriction sites StyI, SpeI, and BamHI (all of them are in red), and primers (dark blue) used for synthesis of the complementary strand and amplification of the product. (C) Schematic of the insertion of 3mer TetO cassette into pSP2 vector through StyI and BamHI sites. Each TetO sequence is indicated with a green box. (D) Schematic of the assembly of the TetO repeat multimers. StyI/SpeI site, created by insertion of the TetO cassette, cannot be cut by either of these two enzymes. (E) Results of the PCRs to amplify 48-mer and 96-mer TetO repeat donors for the HSP70 locus. The expected band sizes were around 3 kb for the 48-mer and around 4.6 kb for the 96-mer TetO donor. NEB 1 kb ladder (M) was used. (F) Schematic of the knock-in strategy. Homology arms (HA) are shown in purple. sgRNA target site is indicated with a red triangle. DSB: double strand break.

    Techniques Used: CRISPR, Knock-In, Polymerase Chain Reaction, Plasmid Preparation, Amplification, Selection, Sequencing

    16) Product Images from "vLIP, a Viral Lipase Homologue, Is a Virulence Factor of Marek's Disease Virus"

    Article Title: vLIP, a Viral Lipase Homologue, Is a Virulence Factor of Marek's Disease Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.11.6984-6996.2005

    Shuttle mutagenesis strategy used to construct vLIP mutant MDVs and revertants in the pRB-1B BAC. A 4.268-kb fragment of the Md5 strain of MDV, containing the vLIP gene and portions of neighboring genes, was cloned into pST76K-SR, a RecA-based shuttle vector. In step 1 (labeled arrow), an in-frame deletion of vLIP amino acids 256 to 426 was incorporated into the pRB-1B BAC by shuttle mutagenesis using a pST76K-SR-based shuttle vector bearing the same deletion (ΔMluI-SpeI), yielding Δ vLIP . In parallel (step 2), an alanine point mutant of the vLIP serine nucleophile position ( vLIP S307A) was incorporated into pRB-1B. As depicted in steps 3 and 4, shuttle mutagenesis was performed on the Δ vLIP BAC to generate C-terminally FLAG tagged vLIP ( vLIP *-rev) and native vLIP ( vLIP -rev) revertants. Relevant features of the DNA fragment used and modified in shuttle mutagenesis procedures are labeled accordingly. A double-headed arrow represents lipase homology in the vLIP ORF, a white X represents the location of the S307A change, a FLAG epitope tag is represented as a labeled asterisk, and the MluI and SpeI sites which were used to remove the lipase homology region of vLIP are labeled accordingly.
    Figure Legend Snippet: Shuttle mutagenesis strategy used to construct vLIP mutant MDVs and revertants in the pRB-1B BAC. A 4.268-kb fragment of the Md5 strain of MDV, containing the vLIP gene and portions of neighboring genes, was cloned into pST76K-SR, a RecA-based shuttle vector. In step 1 (labeled arrow), an in-frame deletion of vLIP amino acids 256 to 426 was incorporated into the pRB-1B BAC by shuttle mutagenesis using a pST76K-SR-based shuttle vector bearing the same deletion (ΔMluI-SpeI), yielding Δ vLIP . In parallel (step 2), an alanine point mutant of the vLIP serine nucleophile position ( vLIP S307A) was incorporated into pRB-1B. As depicted in steps 3 and 4, shuttle mutagenesis was performed on the Δ vLIP BAC to generate C-terminally FLAG tagged vLIP ( vLIP *-rev) and native vLIP ( vLIP -rev) revertants. Relevant features of the DNA fragment used and modified in shuttle mutagenesis procedures are labeled accordingly. A double-headed arrow represents lipase homology in the vLIP ORF, a white X represents the location of the S307A change, a FLAG epitope tag is represented as a labeled asterisk, and the MluI and SpeI sites which were used to remove the lipase homology region of vLIP are labeled accordingly.

    Techniques Used: Mutagenesis, Construct, BAC Assay, Clone Assay, Plasmid Preparation, Labeling, Modification, FLAG-tag

    (a) Location of vLIP relative to other genes in the MDV genome. The vLIP open reading frame is drawn to scale; a bar representing 1 kb is drawn and labeled. The terminal repeat long (TR L ) region, the U L region, and an a-like sequence (double-headed arrow) are also indicated. Several MDV-1-specific ORFs neighboring the vLIP gene are displayed, as are the conserved genes U L 1 to U L 3. The gene organization of the vLIP transcript is shown in more detail below, also drawn to scale. A 500-bp bar is shown, and the TATA box, putatively used to initiate vLIP transcripts, as well as the poly(A) site and poly(A) signal, is labeled. MluI and SpeI restriction sites flanking the lipase homology region, indicated by a double-headed arrow, are also labeled. (b) The vLIP transcript as visualized by Northern blotting. In the left panel, a Northern blot was probed with a single-stranded riboprobe antisense to vLIP mRNA. RNA samples were derived from MSB-1 tumor cells which had been either treated for 24 h in the presence of sodium butyrate to induce virus replication or left untreated, as indicated. PFA was also used where indicated, to show the sensitivity of vLIP transcription to inhibition of DNA replication. To the right, RNA samples used in Northern blotting were separated on denatured agarose gels, stained with ethidium bromide, and imaged to verify that RNA integrity and quantity were comparable across all samples tested. An RNA marker (Ambion) was included to determine molecular weights.
    Figure Legend Snippet: (a) Location of vLIP relative to other genes in the MDV genome. The vLIP open reading frame is drawn to scale; a bar representing 1 kb is drawn and labeled. The terminal repeat long (TR L ) region, the U L region, and an a-like sequence (double-headed arrow) are also indicated. Several MDV-1-specific ORFs neighboring the vLIP gene are displayed, as are the conserved genes U L 1 to U L 3. The gene organization of the vLIP transcript is shown in more detail below, also drawn to scale. A 500-bp bar is shown, and the TATA box, putatively used to initiate vLIP transcripts, as well as the poly(A) site and poly(A) signal, is labeled. MluI and SpeI restriction sites flanking the lipase homology region, indicated by a double-headed arrow, are also labeled. (b) The vLIP transcript as visualized by Northern blotting. In the left panel, a Northern blot was probed with a single-stranded riboprobe antisense to vLIP mRNA. RNA samples were derived from MSB-1 tumor cells which had been either treated for 24 h in the presence of sodium butyrate to induce virus replication or left untreated, as indicated. PFA was also used where indicated, to show the sensitivity of vLIP transcription to inhibition of DNA replication. To the right, RNA samples used in Northern blotting were separated on denatured agarose gels, stained with ethidium bromide, and imaged to verify that RNA integrity and quantity were comparable across all samples tested. An RNA marker (Ambion) was included to determine molecular weights.

    Techniques Used: Labeling, Sequencing, Northern Blot, Derivative Assay, Inhibition, Staining, Marker

    17) Product Images from "Genotypic and Phenotypic Variation in Pseudomonas aeruginosa Reveals Signatures of Secondary Infection and Mutator Activity in Certain Cystic Fibrosis Patients with Chronic Lung Infections ▿ Reveals Signatures of Secondary Infection and Mutator Activity in Certain Cystic Fibrosis Patients with Chronic Lung Infections ▿ †"

    Article Title: Genotypic and Phenotypic Variation in Pseudomonas aeruginosa Reveals Signatures of Secondary Infection and Mutator Activity in Certain Cystic Fibrosis Patients with Chronic Lung Infections ▿ Reveals Signatures of Secondary Infection and Mutator Activity in Certain Cystic Fibrosis Patients with Chronic Lung Infections ▿ †

    Journal: Infection and Immunity

    doi: 10.1128/IAI.05282-11

    Genomic fingerprinting. (A) rep-PCR using BOXA1 primers; (B) SpeI PFGE of SpeI-digested genomic DNA. Percent similarity is indicated on the y axis. *, mutator. Numbers at branch points indicate bootstrap values; only values of > 70 are reported,
    Figure Legend Snippet: Genomic fingerprinting. (A) rep-PCR using BOXA1 primers; (B) SpeI PFGE of SpeI-digested genomic DNA. Percent similarity is indicated on the y axis. *, mutator. Numbers at branch points indicate bootstrap values; only values of > 70 are reported,

    Techniques Used: Polymerase Chain Reaction

    18) Product Images from "Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients"

    Article Title: Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78001-1

    Rolling Circle Amplification (RCA) analysis on patients’ liver biopsies. (a) Workflow of RCA set-up in liver biopsies: DNA extracted from frozen-liver biopsies was first amplified with Phi29 polymerase for 21 h at 30 °C. Amplification products were then either digested with SpeI enzyme and analyzed according to Southern Blot technique using HBV-specific cold probes or assessed following a full-length HBV genomic PCR (P1-P2) followed by gel electrophoresis. (b) Examples of Southern Blot following RCA and SpeI digestion on liver biopsies from patients with different cccDNA concentration measured by qPCR and negative (0; H20) and positive controls (PC; plasmid containing a full-length HBV genome). (c) Examples of gel electrophoresis following full-length HBV genomic PCR (P1-P2) performed on RCA products from patients’ liver biopsies with different cccDNA concentration measured by qPCR and negative (0; H20) and positive control (PC; plasmid containing a full-length HBV genome). MW molecular weight; 0: negative control (H20); PC positive control (plasmid containing a full-length HBV genome).
    Figure Legend Snippet: Rolling Circle Amplification (RCA) analysis on patients’ liver biopsies. (a) Workflow of RCA set-up in liver biopsies: DNA extracted from frozen-liver biopsies was first amplified with Phi29 polymerase for 21 h at 30 °C. Amplification products were then either digested with SpeI enzyme and analyzed according to Southern Blot technique using HBV-specific cold probes or assessed following a full-length HBV genomic PCR (P1-P2) followed by gel electrophoresis. (b) Examples of Southern Blot following RCA and SpeI digestion on liver biopsies from patients with different cccDNA concentration measured by qPCR and negative (0; H20) and positive controls (PC; plasmid containing a full-length HBV genome). (c) Examples of gel electrophoresis following full-length HBV genomic PCR (P1-P2) performed on RCA products from patients’ liver biopsies with different cccDNA concentration measured by qPCR and negative (0; H20) and positive control (PC; plasmid containing a full-length HBV genome). MW molecular weight; 0: negative control (H20); PC positive control (plasmid containing a full-length HBV genome).

    Techniques Used: Amplification, Southern Blot, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Concentration Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Molecular Weight, Negative Control

    19) Product Images from "Gluconobacter as Well as Asaia Species, Newly Emerging Opportunistic Human Pathogens among Acetic Acid Bacteria ▿ Species, Newly Emerging Opportunistic Human Pathogens among Acetic Acid Bacteria ▿ †"

    Article Title: Gluconobacter as Well as Asaia Species, Newly Emerging Opportunistic Human Pathogens among Acetic Acid Bacteria ▿ Species, Newly Emerging Opportunistic Human Pathogens among Acetic Acid Bacteria ▿ †

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00767-10

    PFGE patterns of SpeI-restricted DNAs for the five Gluconobacter sp. strains reported in this study. Lanes 1, 2, and 3, strains aP78, aP81, and aP112 (case 2), respectively; lane 4, strain aP90 (case 3); lane 5, strain LBN 175 (case 1); lanes Sc and
    Figure Legend Snippet: PFGE patterns of SpeI-restricted DNAs for the five Gluconobacter sp. strains reported in this study. Lanes 1, 2, and 3, strains aP78, aP81, and aP112 (case 2), respectively; lane 4, strain aP90 (case 3); lane 5, strain LBN 175 (case 1); lanes Sc and

    Techniques Used:

    20) Product Images from "Increased PHGDH expression uncouples hair follicle cycle progression and promotes inappropriate melanin accumulation"

    Article Title: Increased PHGDH expression uncouples hair follicle cycle progression and promotes inappropriate melanin accumulation

    Journal: bioRxiv

    doi: 10.1101/249250

    Generation of the PHGDH tetO allele (A) Schematic of the Col1A locus in wildtype mouse cells (top), the modified locus in KH2 ES cells (middle) and the locus after targeting to introduce the PHGDH tetO allele (bottom). The PHGDH cDNA introduced into the Col1A locus is the human sequence. The expected band sizes when the indicated probe is used for Southern blot analysis as in (B) and (C) ] are indicated. Also shown are the location of the SpeI sites (marked “S”) in each locus used to digest genomic DNA for Southern blot analysis. FRT, flippase recognition target site; tetO, tetracycline operator minimal promoter. (B) Southern blot analysis of SpeI-digested genomic DNA from six PHGDH tetO -targeted ES cells. Clones D4 and D5 exhibit proper targeting of the Col1A locus and an unaffected wild-type allele. (C) Southern blot analysis of SpeI-digested genomic DNA from mice of the indicated genotypes. (D) PCR-based genotyping of the PHGDH tetO and Rosa26-M2rtTA alleles. In the PHGDH tetO reaction, the presence of the transgene is indicated by the upper band; in the Rosa26-M2rtTA reaction, the presence of the transgene is indicated by the lower band. (E) The number of offspring of each genotype observed when mice hemizygous for the PHGDH tetO allele exposed to a doxycycline diet were mated. The observed distribution of genotypes in the offspring did not differ significantly from expected Mendelian ratios, with p=0.58 by the χ 2 goodness-of-fit test. (F) Western blot analysis of PHGDH protein using the indicated amount of recombinant human or mouse PHGDH to test antibody specificity.
    Figure Legend Snippet: Generation of the PHGDH tetO allele (A) Schematic of the Col1A locus in wildtype mouse cells (top), the modified locus in KH2 ES cells (middle) and the locus after targeting to introduce the PHGDH tetO allele (bottom). The PHGDH cDNA introduced into the Col1A locus is the human sequence. The expected band sizes when the indicated probe is used for Southern blot analysis as in (B) and (C) ] are indicated. Also shown are the location of the SpeI sites (marked “S”) in each locus used to digest genomic DNA for Southern blot analysis. FRT, flippase recognition target site; tetO, tetracycline operator minimal promoter. (B) Southern blot analysis of SpeI-digested genomic DNA from six PHGDH tetO -targeted ES cells. Clones D4 and D5 exhibit proper targeting of the Col1A locus and an unaffected wild-type allele. (C) Southern blot analysis of SpeI-digested genomic DNA from mice of the indicated genotypes. (D) PCR-based genotyping of the PHGDH tetO and Rosa26-M2rtTA alleles. In the PHGDH tetO reaction, the presence of the transgene is indicated by the upper band; in the Rosa26-M2rtTA reaction, the presence of the transgene is indicated by the lower band. (E) The number of offspring of each genotype observed when mice hemizygous for the PHGDH tetO allele exposed to a doxycycline diet were mated. The observed distribution of genotypes in the offspring did not differ significantly from expected Mendelian ratios, with p=0.58 by the χ 2 goodness-of-fit test. (F) Western blot analysis of PHGDH protein using the indicated amount of recombinant human or mouse PHGDH to test antibody specificity.

    Techniques Used: Modification, Introduce, Sequencing, Southern Blot, Clone Assay, Mouse Assay, Polymerase Chain Reaction, Genotyping Assay, Western Blot, Recombinant

    21) Product Images from "Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus"

    Article Title: Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus

    Journal: Viruses

    doi: 10.3390/v13112119

    ( a ) Strategy for the construction of a full-length DNA-launched infectious clone of PAstV. The T7 promoter of pMD123 was replaced with a cytomegalovirus (CMV) promotor using SpeI and NgoMIV . The hepatitis delta virus ribozyme was inserted into the 3′ terminal end of the PAstV-GX1 genome with XhoI and SacII. The restriction enzyme sites above the full-length cDNA clones are indicated by arrows. ( b ) Cytopathic effects were observed in PK-15 cells infected with rescued viruses (magnification 10×). ( c ) IFA analysis of the Cap protein expression in PK-15 cells. PK-15 cells infected with rescued and parental viruses were fixed at approximately 48 h.p.i and subjected to immunofluorescence using a polyclonal antibody against capsid protein. The PK-15 cells were stained with goat anti-mouse IgG H L (CoraLite488) (magnification 20×). ( d ) Growth curves of the recombinant and parental viruses. PK-15 cells were infected with recovered and parental viruses at a multiplicity of infection of 0.01. The viral titers were determined as TCID 50 .
    Figure Legend Snippet: ( a ) Strategy for the construction of a full-length DNA-launched infectious clone of PAstV. The T7 promoter of pMD123 was replaced with a cytomegalovirus (CMV) promotor using SpeI and NgoMIV . The hepatitis delta virus ribozyme was inserted into the 3′ terminal end of the PAstV-GX1 genome with XhoI and SacII. The restriction enzyme sites above the full-length cDNA clones are indicated by arrows. ( b ) Cytopathic effects were observed in PK-15 cells infected with rescued viruses (magnification 10×). ( c ) IFA analysis of the Cap protein expression in PK-15 cells. PK-15 cells infected with rescued and parental viruses were fixed at approximately 48 h.p.i and subjected to immunofluorescence using a polyclonal antibody against capsid protein. The PK-15 cells were stained with goat anti-mouse IgG H L (CoraLite488) (magnification 20×). ( d ) Growth curves of the recombinant and parental viruses. PK-15 cells were infected with recovered and parental viruses at a multiplicity of infection of 0.01. The viral titers were determined as TCID 50 .

    Techniques Used: Clone Assay, Infection, Immunofluorescence, Expressing, Staining, Recombinant

    22) Product Images from "Defining natural species of bacteria: clear-cut genomic boundaries revealed by a turning point in nucleotide sequence divergence"

    Article Title: Defining natural species of bacteria: clear-cut genomic boundaries revealed by a turning point in nucleotide sequence divergence

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-489

    Genomic DNA PFGE patterns of representative Salmonella strains, cleaved with I-CeuI (a), XbaI (b) and SpeI (c), respectively. Lanes: 1, DNA size Marker; 2-5, S. enteritidis (SE310, SE154, SE301, LK5); 6-14, S. pullorum (RKS5078, CDC1983-67, SARB51, 04–6767, NS387, 00–19557, 02–15951, 03–16062, 98–13777); 15–20, S. gallinarum (287/91, RKS5021, SGSC2293, 91–29327, 90–5289, 92–7995).
    Figure Legend Snippet: Genomic DNA PFGE patterns of representative Salmonella strains, cleaved with I-CeuI (a), XbaI (b) and SpeI (c), respectively. Lanes: 1, DNA size Marker; 2-5, S. enteritidis (SE310, SE154, SE301, LK5); 6-14, S. pullorum (RKS5078, CDC1983-67, SARB51, 04–6767, NS387, 00–19557, 02–15951, 03–16062, 98–13777); 15–20, S. gallinarum (287/91, RKS5021, SGSC2293, 91–29327, 90–5289, 92–7995).

    Techniques Used: Marker

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    New England Biolabs spei
    OSN-specific GABAR B1 and D2R knockout mice, Related to Figure 2 . (A) Gene targeting at Drd2 locus. We obtained germline transmission from one ES clone, HEPD064_5_D11. The Drd2 locus in Drd2 tm1a , Drd2 fl (= Drd tm1c ) and Drd cKO . Drd2 +/ tm1a mice were crossed with Flp mice to obtain Drd2 +/ fl . ( C ) Gene targeting at Gabbr1 locus. We obtained germline transmission from one ES clone, EPD0730_1_G04. ( D ) The Gabbr1 locus in Gabbr1 tm1a , Gabbr1 fl (= Gabbr1 tm1c ), and Gabbr1 cKO . Gabbr1 +/ tm1a mice were crossed with Flp mice to obtain Gabbr1 +/ fl . ( E-G ) Southern blot analysis on Drd2 +/+ and Drd2 tm1a /+ ( E , Drd2 probe), Gabbr1 +/+ and Gabbr1 tm1a /+ ( F , Gabbr1 probe); Drd2 tm1a /+ and Gabbr1 tm1a /+ ( G , neo r probe). ( H ) Immunostaining of D2R in the OB of OSN-specific Drd2 mutant mice. ( I ) Immunostaining of GABAR B1 in the OB of OSN-specific Gabbr1 mutant mice. Location of 5’ and 3’ arms for gene targeting and 5’ and 3’ DNA probes for southern blotting are shown in ( A ) and ( C ). <t>KpnI</t> (K), <t>SpeI</t> (S), XhoI (X) and ApaI (A).
    Spei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs spei restriction enzymes
    Plasmid stability. GFP expression was induced with 500 ng/ml of ATc. ON culture of day 0 was supplemented with 15 μg/ml kanamycin, while ON cultures of days 1–4 were supplemented with 50 μg/ml ampicillin. (A) GFP expression in F. novicida U112 pFNMB1 msfgfp ON cultures of day 0 and day 4. (B) GFP expression in F. novicida U112 pFNMB2 msfgfp ON cultures of day 0 and day 4. (A,B) Images are a merge of phase contrast and GFP channel. 26 × 39 μm fields of view are shown. Scale bar represent 5 μm. Representative replicates are shown. Three independent experiments were performed. (C) Survival assay performed with ON cultures of F. novicida U112 pFNMB1 msfgfp and F. novicida U112 pFNMB2 msfgfp plated on ampicillin and ampicillin/kanamycin plates. Three independent experiments were performed. (D) Digestion of pFNMB1 msfgfp with <t>SacI</t> and <t>SpeI</t> restriction enzymes. (E) Digestion of pFNMB2 msfgfp with SacI and SpeI restriction enzymes. (D,E) Plasmids were passaged in F. novicida for 4 days before being transformed into E. coli . Controls were isolated directly from E. coli . Representative replicates are shown. Three independent experiments were performed.
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    New England Biolabs spei hf
    Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and <t>MfeI-HF),</t> green = four restriction enzymes (KpnI-HF, Mfe-HFI, <t>SpeI-HF,</t> EcoRV-HF)).
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    OSN-specific GABAR B1 and D2R knockout mice, Related to Figure 2 . (A) Gene targeting at Drd2 locus. We obtained germline transmission from one ES clone, HEPD064_5_D11. The Drd2 locus in Drd2 tm1a , Drd2 fl (= Drd tm1c ) and Drd cKO . Drd2 +/ tm1a mice were crossed with Flp mice to obtain Drd2 +/ fl . ( C ) Gene targeting at Gabbr1 locus. We obtained germline transmission from one ES clone, EPD0730_1_G04. ( D ) The Gabbr1 locus in Gabbr1 tm1a , Gabbr1 fl (= Gabbr1 tm1c ), and Gabbr1 cKO . Gabbr1 +/ tm1a mice were crossed with Flp mice to obtain Gabbr1 +/ fl . ( E-G ) Southern blot analysis on Drd2 +/+ and Drd2 tm1a /+ ( E , Drd2 probe), Gabbr1 +/+ and Gabbr1 tm1a /+ ( F , Gabbr1 probe); Drd2 tm1a /+ and Gabbr1 tm1a /+ ( G , neo r probe). ( H ) Immunostaining of D2R in the OB of OSN-specific Drd2 mutant mice. ( I ) Immunostaining of GABAR B1 in the OB of OSN-specific Gabbr1 mutant mice. Location of 5’ and 3’ arms for gene targeting and 5’ and 3’ DNA probes for southern blotting are shown in ( A ) and ( C ). KpnI (K), SpeI (S), XhoI (X) and ApaI (A).

    Journal: bioRxiv

    Article Title: Widespread inhibition, antagonism, and synergy in mouse olfactory sensory neurons in vivo

    doi: 10.1101/803908

    Figure Lengend Snippet: OSN-specific GABAR B1 and D2R knockout mice, Related to Figure 2 . (A) Gene targeting at Drd2 locus. We obtained germline transmission from one ES clone, HEPD064_5_D11. The Drd2 locus in Drd2 tm1a , Drd2 fl (= Drd tm1c ) and Drd cKO . Drd2 +/ tm1a mice were crossed with Flp mice to obtain Drd2 +/ fl . ( C ) Gene targeting at Gabbr1 locus. We obtained germline transmission from one ES clone, EPD0730_1_G04. ( D ) The Gabbr1 locus in Gabbr1 tm1a , Gabbr1 fl (= Gabbr1 tm1c ), and Gabbr1 cKO . Gabbr1 +/ tm1a mice were crossed with Flp mice to obtain Gabbr1 +/ fl . ( E-G ) Southern blot analysis on Drd2 +/+ and Drd2 tm1a /+ ( E , Drd2 probe), Gabbr1 +/+ and Gabbr1 tm1a /+ ( F , Gabbr1 probe); Drd2 tm1a /+ and Gabbr1 tm1a /+ ( G , neo r probe). ( H ) Immunostaining of D2R in the OB of OSN-specific Drd2 mutant mice. ( I ) Immunostaining of GABAR B1 in the OB of OSN-specific Gabbr1 mutant mice. Location of 5’ and 3’ arms for gene targeting and 5’ and 3’ DNA probes for southern blotting are shown in ( A ) and ( C ). KpnI (K), SpeI (S), XhoI (X) and ApaI (A).

    Article Snippet: Southern blotting Genomic DNA (10 μg) extracted from the mouse tail was digested with restriction enzymes: KpnI (Toyobo), ApaI (NEB, R0114S), SpeI (NEB, R0133S), and XhoI (NEB, R0146S).

    Techniques: Knock-Out, Mouse Assay, Transmission Assay, Southern Blot, Immunostaining, Mutagenesis

    Plasmid stability. GFP expression was induced with 500 ng/ml of ATc. ON culture of day 0 was supplemented with 15 μg/ml kanamycin, while ON cultures of days 1–4 were supplemented with 50 μg/ml ampicillin. (A) GFP expression in F. novicida U112 pFNMB1 msfgfp ON cultures of day 0 and day 4. (B) GFP expression in F. novicida U112 pFNMB2 msfgfp ON cultures of day 0 and day 4. (A,B) Images are a merge of phase contrast and GFP channel. 26 × 39 μm fields of view are shown. Scale bar represent 5 μm. Representative replicates are shown. Three independent experiments were performed. (C) Survival assay performed with ON cultures of F. novicida U112 pFNMB1 msfgfp and F. novicida U112 pFNMB2 msfgfp plated on ampicillin and ampicillin/kanamycin plates. Three independent experiments were performed. (D) Digestion of pFNMB1 msfgfp with SacI and SpeI restriction enzymes. (E) Digestion of pFNMB2 msfgfp with SacI and SpeI restriction enzymes. (D,E) Plasmids were passaged in F. novicida for 4 days before being transformed into E. coli . Controls were isolated directly from E. coli . Representative replicates are shown. Three independent experiments were performed.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida

    doi: 10.3389/fcimb.2018.00284

    Figure Lengend Snippet: Plasmid stability. GFP expression was induced with 500 ng/ml of ATc. ON culture of day 0 was supplemented with 15 μg/ml kanamycin, while ON cultures of days 1–4 were supplemented with 50 μg/ml ampicillin. (A) GFP expression in F. novicida U112 pFNMB1 msfgfp ON cultures of day 0 and day 4. (B) GFP expression in F. novicida U112 pFNMB2 msfgfp ON cultures of day 0 and day 4. (A,B) Images are a merge of phase contrast and GFP channel. 26 × 39 μm fields of view are shown. Scale bar represent 5 μm. Representative replicates are shown. Three independent experiments were performed. (C) Survival assay performed with ON cultures of F. novicida U112 pFNMB1 msfgfp and F. novicida U112 pFNMB2 msfgfp plated on ampicillin and ampicillin/kanamycin plates. Three independent experiments were performed. (D) Digestion of pFNMB1 msfgfp with SacI and SpeI restriction enzymes. (E) Digestion of pFNMB2 msfgfp with SacI and SpeI restriction enzymes. (D,E) Plasmids were passaged in F. novicida for 4 days before being transformed into E. coli . Controls were isolated directly from E. coli . Representative replicates are shown. Three independent experiments were performed.

    Article Snippet: The plasmids were recovered again, digested with SacI and SpeI restriction enzymes and loaded on an agarose gel to analyze the size of the DNA fragments.

    Techniques: Plasmid Preparation, Expressing, Clonogenic Cell Survival Assay, Transformation Assay, Isolation

    Plasmid stability. GFP expression was induced with 500 ng/ml of ATc. ON culture of day 0 was supplemented with 15 μg/ml kanamycin, while ON cultures of days 1–4 were supplemented with 50 μg/ml ampicillin. (A) GFP expression in F. novicida U112 pFNMB1 msfgfp ON cultures of day 0 and day 4. (B) GFP expression in F. novicida U112 pFNMB2 msfgfp ON cultures of day 0 and day 4. (A,B) Images are a merge of phase contrast and GFP channel. 26 × 39 μm fields of view are shown. Scale bar represent 5 μm. Representative replicates are shown. Three independent experiments were performed. (C) Survival assay performed with ON cultures of F. novicida U112 pFNMB1 msfgfp and F. novicida U112 pFNMB2 msfgfp plated on ampicillin and ampicillin/kanamycin plates. Three independent experiments were performed. (D) Digestion of pFNMB1 msfgfp with SacI and SpeI restriction enzymes. (E) Digestion of pFNMB2 msfgfp with SacI and SpeI restriction enzymes. (D,E) Plasmids were passaged in F. novicida for 4 days before being transformed into E. coli . Controls were isolated directly from E. coli . Representative replicates are shown. Three independent experiments were performed.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida

    doi: 10.3389/fcimb.2018.00284

    Figure Lengend Snippet: Plasmid stability. GFP expression was induced with 500 ng/ml of ATc. ON culture of day 0 was supplemented with 15 μg/ml kanamycin, while ON cultures of days 1–4 were supplemented with 50 μg/ml ampicillin. (A) GFP expression in F. novicida U112 pFNMB1 msfgfp ON cultures of day 0 and day 4. (B) GFP expression in F. novicida U112 pFNMB2 msfgfp ON cultures of day 0 and day 4. (A,B) Images are a merge of phase contrast and GFP channel. 26 × 39 μm fields of view are shown. Scale bar represent 5 μm. Representative replicates are shown. Three independent experiments were performed. (C) Survival assay performed with ON cultures of F. novicida U112 pFNMB1 msfgfp and F. novicida U112 pFNMB2 msfgfp plated on ampicillin and ampicillin/kanamycin plates. Three independent experiments were performed. (D) Digestion of pFNMB1 msfgfp with SacI and SpeI restriction enzymes. (E) Digestion of pFNMB2 msfgfp with SacI and SpeI restriction enzymes. (D,E) Plasmids were passaged in F. novicida for 4 days before being transformed into E. coli . Controls were isolated directly from E. coli . Representative replicates are shown. Three independent experiments were performed.

    Article Snippet: The plasmids were recovered again, digested with SacI and SpeI restriction enzymes and loaded on an agarose gel to analyze the size of the DNA fragments.

    Techniques: Plasmid Preparation, Expressing, Clonogenic Cell Survival Assay, Transformation Assay, Isolation

    Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and MfeI-HF), green = four restriction enzymes (KpnI-HF, Mfe-HFI, SpeI-HF, EcoRV-HF)).

    Journal: bioRxiv

    Article Title: Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

    doi: 10.1101/203919

    Figure Lengend Snippet: Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and MfeI-HF), green = four restriction enzymes (KpnI-HF, Mfe-HFI, SpeI-HF, EcoRV-HF)).

    Article Snippet: To improve the observed on-target capture rate, a genome complexity reduction step was added before library preparation by pre-digesting genomic DNA samples with high fidelity restriction enzymes, KpnI-HF, MfeI-HF, SpeI-HF, and EcoRV-HF (New England Biolabs), in the presence of calf intestinal alkaline phosphatase (CIP, New England Biolabs).

    Techniques: