xbai Promega Search Results


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  • 99
    New England Biolabs xbai
    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in <t>pUC57-kan</t> vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and <t>XbaI</t> restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.
    Xbai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lipofectamine 2000
    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in <t>pUC57-kan</t> vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and <t>XbaI</t> restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 466214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega pgl3 control vector
    PTEN and SMAD7 were the direct target genes of miR-216a. (A) In HCF cells, the relative luciferase activity was found to be significantly decreased when <t>pGL3-PTEN-3’-UTR</t> or pGL3-SMAD7-3’-UTR was transfected together with miR-216a mimic but not with the miRNA mimic control; (B) 72 hrs after the transfection in HCF cells, western blot revealed that the protein expression levels of PTEN and SMAD7 were significantly down-regulated in cells transfected with miR-216a mimic relative to those transfected with miRNA mimic control. *P
    Pgl3 Control Vector, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 8028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pgem t easy vector
    Strategies for generation of dsRNAs and d-siRNAs. (A) <t>PCR</t> template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.
    Pgem T Easy Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 91938 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Promega pgl3 basic vector
    miR-144 regulates agrin expression by directly targeting the 3′ UTR of agrin mRNA. (A) Schematic representation of three putative miRNA-binding sites on the agrin 3′ UTR sequence. (B) The 3′ UTR fragment of agrin was cloned into the Xbal restriction site downstream of the firefly luciferase gene of the <t>pGL3-Basic</t> vector. Luciferase activity of each sample was measured 48h after transfection and normalized to Renilla luciferase activity. pGL3-Basic vector was used as control. Graph error bars indicate SD calculated from at least three independent experiments. * P
    Pgl3 Basic Vector, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 28647 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega pmirglo dual luciferase mirna target expression vector
    Reporter gene assay for the functional analysis of 3′-UTR regulated by <t>miRNA.</t> ( A ) The reporter construct contained the inserted DNA fragment of human SLC30A3 3′-UTR wild type (WT) or mutant (Mut) downstream of luc2 in the <t>pmirGLO</t> Dual-Luciferase miRNA Target Expression Vector. ( B ) Reporter gene assay was performed using CHO cells co-transfected with NC or miR-5572 mimic, and the reporter vector cloned with WT 3′-UTR of SLC30A3 or mutant 3′-UTR of SLC30A3 ( n = 3 in each experimental group). All data are presented as box and scatter plot. Statistical significance was determined by two-way ANOVA followed by post hoc Bonferroni’s test (* p
    Pmirglo Dual Luciferase Mirna Target Expression Vector, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 6909 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega pmirglo vector
    miRNA-29c-3p directly binds in the 3′UTR of AKT3, TGIF2, CREB5, and CDK6 A . Target Scan predicted base pairing of mature miRNA-29c-3p seed sequences in the 3′UTRs of the indicated genes. B . Luciferase activity in MCF10.AT1 cells transfected with the dual luciferase vector <t>pmiRGLo</t> containing the wild type (Wt) and mutated (Mut) miRNA-29c-3p binding sites indicated in panel A. The cells were also transfected with an miRNA-29c-3p mimic or a negative control mimic. The firefly signal reported the gene regulation by miRNA binding and the internal control renilla luciferase was used for normalizing the transfection efficiency. * p
    Pmirglo Vector, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 4939 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega xba i
    Pulsed field gel electrophoresis <t>(PFGE)</t> types for Xba I-digested genomic DNA of Salmonella enterica strains isolated from raw chicken meat Four different PFGE types ( A, B, C, D ) were generated according to serotypes. M – λ ladder marker for PFGE; ATCC – Salmonella Typhimurium ATCC 19585 control strain; LMM300 – S . Meleagridis; LMM288 – S . Heidelberg; LMM218 – S . Enteritidis; LMM182 – S . Heidelberg; LMM179 – S . Heidelberg; LMM175 – S . Heidelberg; LMM170 – S . Enteritidis; LMM120 – S . Typhimurium; LMM105 – S . Heidelberg.
    Xba I, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 2188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega pgl3 basic
    Cell cycle-regulated expression of the CDC6 promoter is dependent on E2F DNA-binding sites. NIH 3T3 cells were transiently transfected with p[−130,+225], <t>pGL3-DM,</t> p[−570,+98], and p[−266,+98]. Forty-eight hours posttransfection, cells were serum starved for 24 h and subsequently stimulated with fresh medium containing 10% BCS. Lysates were made after the depicted time spans. Asynchronous (A) samples were valued as 100 adjusted luciferase counts, while the others were calculated in comparison to this. Transfection efficiencies were determined by pCMV-βgal cotransfection. Samples were obtained in duplicate, and the presented data are representative for at least three independent experiments.
    Pgl3 Basic, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 12868 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega t7 rna polymerase
    A codon-modified GAr sequence influences EBNA1 synthesis. ( A ) IVT assay of pcDNA3 expression constructs encoding EBNA1 (E1) (lane 1), E1ΔGA (lane 2), E1-GAr(100N) (lane 3), E1-GAr(100M) (lane 4), E1-GAr(200N) (lane 5), E1-GAr(200M) (lane 6), E1-GAr(300N) (lane 7), E1-GAr(300M) (lane 8), E1-GAr(400N) (lane 9), E1-GAr(400M) (lane 10), E1-GAr(500N) (lane 11), or E1-GAr(500M) (lane 12). The constructs were transcribed and translated in vitro with <t>T7</t> RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. 35 S-methionine-labeled proteins were visualized by autoradiography. ( B and C ) Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities ( B ) or relative fold increase of EBNA1 encoded by codon-modified GAr domains compared with EBNA1 encoded by native GAr domains ( C ). ( D ) Western blot of EBV-negative HEK293 cells transfected with expression constructs encoding E1-GFP (lane 1), E1ΔGA-GFP (lane 2), E1-GAr(100N)-GFP (lane 3), E1-GAr(100M)-GFP (lane 4), E1-GAr(200N)-GFP (lane 5), E1-GAr(200M)-GFP (lane 6), E1-GAr(300N)-GFP (lane 7), E1-GAr(300M)-GFP (lane 8), E1-GAr(400N)-GFP (lane 9), E1-GAr(400M)-GFP (lane 10), E1-GAr(500N)-GFP (lane 11), or E1-GAr(500M)-GFP (lane 12) with a GFP antibody ( Upper ) or a monoclonal actin antibody ( Lower ). Molecular weight markers M r (kDa) are indicated on the left. ( E ) Band intensities after immunoblotting were quantified as described for B . Representative data from one of four experiments are presented here.
    T7 Rna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 10131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pgem t vector
    Restriction maps of the pAX1 series vectors. The parental plasmid pAX1 is a derivative of <t>pGEM-T</t> (Table ), which contains a unique XbaI site that is flanked by DNA homologous to the intergenic region between PD702 and PD703. The DNA
    Pgem T Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 30613 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega t4 dna ligase
    Restriction maps of the pAX1 series vectors. The parental plasmid pAX1 is a derivative of <t>pGEM-T</t> (Table ), which contains a unique XbaI site that is flanked by DNA homologous to the intergenic region between PD702 and PD703. The DNA
    T4 Dna Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 11518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    luc  (Promega)
    99
    Promega luc
    Involvement of Ago2, TRBP2 and PACT in modified-siRNA-dependent gene silencing. ( A ) PACT and TRBP2 binding to siRNA (siLuc-36) and cognate siDNA. The 5′end of the guide strand of siRNA or siDNA was phosphorylated with [γ- 32 P] ATP and the 5′end of the passenger strand was phosphorylated with cold ATP. A mobility shift assay was carried out using PACT and TRBP2 proteins purified from E. coli cells. Note that TRBP2 and PACT are capable of binding to dsRNA but not dsDNA. Arrows indicate the position of protein–nucleic acid complexes. ( B ) Reduced expression of Ago2 and TRBP2 by RNAi. HeLa cells were transfected with siGY441 (control siRNA), siAgo2 or siTRBP2 (50 nM) along with pCAGIPuro-EGFP (0.5 µg) and subjected to RT–PCR 3 days after transfection. Note that Ago2 and TRBP2 RNA were specifically knocked down by siAgo2 and siTRBP2, respectively. ( C ) Requirement of Ago2 and TRBP2 for gene silencing due to transfection with modified siRNA with 8-bp GS-DNA substitution. HeLa cells were simultaneously transfected with <t>pGL3-Control</t> DNA (1 µg) and pRL-SV40 DNA (0.1 µg), and a mixture of siRNA and modified siRNA with DNA substitution. Two siRNAs, siLuc-36 (left panel) and siLuc-774 (right panel), and their modified siRNA counterparts, 5 nM each, were used for <t>luc</t> gene silencing. chiLuc-36 and chiLuc-774-GS are functional DNA-modified siRNAs specific to luc knockdown, whereas chiLuc-774-PS is a modified siRNA with DNA replacement in the PS-end-proximal region. Ago2 and TRBP2 were knocked down using 50 nM siAgo2 and siTRBP2, respectively. As an siRNA concentration control, siGY441 (50 nM), an siRNA for EGFP knockdown, was used. Gene-silencing activity was measured 24 h after transfection. Note both nonmodified and DNA-modified siRNA-dependent luc gene-silencing activity to be reduced by 30–50 points by knocking down Ago2 and TRBP2 activity through siAgo2 and siTRBP2 RNAi, respectively. ( D ) Functional DNA-modified siRNA-dependent target mRNA cleavage. HeLa cells were cotransfected with pTREC-2-153 and siLuc-1085 (a negative control siRNA), siLuc2-153 (target-cleavable siRNA) or its cognate modified siRNA with 8-bp GS-DNA substitution, each 5 nM. RNA was extracted 24 h after transfection, and cleavage sites were determined by primer extension. Sequence ladder was prepared using the same pTREC construct ( 25 ). Note that the position of the main cleavage site is precisely identical between RNAi and gene silencing due to transfection with modified siRNA with 8-bp GS-DNA substitution.
    Luc, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1660 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Stratagene quikchange site directed mutagenesis kit
    Involvement of Ago2, TRBP2 and PACT in modified-siRNA-dependent gene silencing. ( A ) PACT and TRBP2 binding to siRNA (siLuc-36) and cognate siDNA. The 5′end of the guide strand of siRNA or siDNA was phosphorylated with [γ- 32 P] ATP and the 5′end of the passenger strand was phosphorylated with cold ATP. A mobility shift assay was carried out using PACT and TRBP2 proteins purified from E. coli cells. Note that TRBP2 and PACT are capable of binding to dsRNA but not dsDNA. Arrows indicate the position of protein–nucleic acid complexes. ( B ) Reduced expression of Ago2 and TRBP2 by RNAi. HeLa cells were transfected with siGY441 (control siRNA), siAgo2 or siTRBP2 (50 nM) along with pCAGIPuro-EGFP (0.5 µg) and subjected to RT–PCR 3 days after transfection. Note that Ago2 and TRBP2 RNA were specifically knocked down by siAgo2 and siTRBP2, respectively. ( C ) Requirement of Ago2 and TRBP2 for gene silencing due to transfection with modified siRNA with 8-bp GS-DNA substitution. HeLa cells were simultaneously transfected with <t>pGL3-Control</t> DNA (1 µg) and pRL-SV40 DNA (0.1 µg), and a mixture of siRNA and modified siRNA with DNA substitution. Two siRNAs, siLuc-36 (left panel) and siLuc-774 (right panel), and their modified siRNA counterparts, 5 nM each, were used for <t>luc</t> gene silencing. chiLuc-36 and chiLuc-774-GS are functional DNA-modified siRNAs specific to luc knockdown, whereas chiLuc-774-PS is a modified siRNA with DNA replacement in the PS-end-proximal region. Ago2 and TRBP2 were knocked down using 50 nM siAgo2 and siTRBP2, respectively. As an siRNA concentration control, siGY441 (50 nM), an siRNA for EGFP knockdown, was used. Gene-silencing activity was measured 24 h after transfection. Note both nonmodified and DNA-modified siRNA-dependent luc gene-silencing activity to be reduced by 30–50 points by knocking down Ago2 and TRBP2 activity through siAgo2 and siTRBP2 RNAi, respectively. ( D ) Functional DNA-modified siRNA-dependent target mRNA cleavage. HeLa cells were cotransfected with pTREC-2-153 and siLuc-1085 (a negative control siRNA), siLuc2-153 (target-cleavable siRNA) or its cognate modified siRNA with 8-bp GS-DNA substitution, each 5 nM. RNA was extracted 24 h after transfection, and cleavage sites were determined by primer extension. Sequence ladder was prepared using the same pTREC construct ( 25 ). Note that the position of the main cleavage site is precisely identical between RNAi and gene silencing due to transfection with modified siRNA with 8-bp GS-DNA substitution.
    Quikchange Site Directed Mutagenesis Kit, supplied by Stratagene, used in various techniques. Bioz Stars score: 95/100, based on 66977 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pcdna3 1
    Gene expression efficacies of MENDs in BMDCs. ( a ) Schematic diagram of the R8-MEND (left) and KALA-MEND (right). ( b ) The R8-MEND and KALA-MEND encapsulating a conventional pDNA <t>(pcDNA3.1-Luc;</t> opened bar) or CpG-free pDNA (pCpGfree-Luc(0); closed bar) were transfected to BMDCs. Data were presented as the mean ± SD of three independent experiments. Statistical differences were evaluated by one-way ANOVA, followed by Student's t -test (** P
    Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 49697 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega pfu dna polymerase
    Gene expression efficacies of MENDs in BMDCs. ( a ) Schematic diagram of the R8-MEND (left) and KALA-MEND (right). ( b ) The R8-MEND and KALA-MEND encapsulating a conventional pDNA <t>(pcDNA3.1-Luc;</t> opened bar) or CpG-free pDNA (pCpGfree-Luc(0); closed bar) were transfected to BMDCs. Data were presented as the mean ± SD of three independent experiments. Statistical differences were evaluated by one-way ANOVA, followed by Student's t -test (** P
    Pfu Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 6001 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega human genomic dna
    miR-15b-5p, predicted to be induced in DFUs, and suppression of its targets, <t>DNA</t> repair and inflammatory regulatory genes. a. IPA identified miR-15b-5p as the top enriched miR predicted to target DFU regulated genes from microarrays. b. miR-15b-5p predicted targets comprise regulators of the inflammatory response and genes involved in DNA repair. c. IKBKB and <t>WEE1,</t> are down-regulated in HaCaT cells transfected with mimic miR-15b-5p. MSH2 and RAD50 showed a trend of down-regulation but did not reach statistical significance. ctrl = mimic negative control #1. d. IKBKB and WEE1 are down-regulated in S. aureus infected human ex-vivo wounds in comparison to control un-infected wounds. (n=3 independent experiments, paired t-test was used to determine statistical significance, * = p-value
    Human Genomic Dna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 7262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Journal: Scientific Reports

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli

    doi: 10.1038/s41598-018-30792-0

    Figure Lengend Snippet: pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Article Snippet: For the simultaneous expression of Nampt and PRPP synthetase a bicistronic vector was constructed by PCR amplifying the baPrs sequence (Q5 High-Fidelity 2X Master Mix, NEB) from pUC57-Kan plasmid with M13 forward and reverse primers, double digestion with XbaI and NcoI restriction enzymes (NEB) of PCR product and pET28a-hdNAdV plasmid, followed by the separation of the desired DNA fragments on 1.5% agarose gel electrophoresis , purification from gel (using Wizard SV Gel and PCR Clean-Up System, Promega, Madison, USA) of desired bands (pET28a-hdNadV and baPrs) followed by ligation with T4 DNA ligase (NEB) (as shown in Fig. ).

    Techniques: Plasmid Preparation, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Amplification, Purification, Construct, Transformation Assay, Agarose Gel Electrophoresis, Fluorimetry Assay

    Estimation of C. felis 18S rDNA copy number using Southern blot. C. felis genomic DNA (6 µg/enzyme) was digested with Eag I, Eco R I, Pst I, Xba I and Xho I. Uncut gDNA served as a negative control and PCR product of a portion of C. felis 18S rDNA served as a positive control. Genomic DNA was hybridized with Cf 18S bp probe to estimate the number of 18S rDNA gene copies in C. felis . Digestion with each enzyme results in a single digestion product. (*DNA was not completely digested by Pst I. Top band is uncut DNA.)

    Journal: PLoS ONE

    Article Title: Prevalence and Infection Load Dynamics of Rickettsia felis in Actively Feeding Cat Fleas

    doi: 10.1371/journal.pone.0002805

    Figure Lengend Snippet: Estimation of C. felis 18S rDNA copy number using Southern blot. C. felis genomic DNA (6 µg/enzyme) was digested with Eag I, Eco R I, Pst I, Xba I and Xho I. Uncut gDNA served as a negative control and PCR product of a portion of C. felis 18S rDNA served as a positive control. Genomic DNA was hybridized with Cf 18S bp probe to estimate the number of 18S rDNA gene copies in C. felis . Digestion with each enzyme results in a single digestion product. (*DNA was not completely digested by Pst I. Top band is uncut DNA.)

    Article Snippet: Briefly, gDNA was extracted from Rickettsia -uninfected, unfed adult cat fleas (Heska Corporation, Loveland, CO) using Qiagen DNeasy DNA Extraction Kit and digested with five restriction enzymes (6 µg gDNA/enzyme): Eag I, Pst I , Xba I, Xho I (New England BioLabs, Ipswich, MA), and Eco R I (Promega, Madison, WI).

    Techniques: Southern Blot, Negative Control, Polymerase Chain Reaction, Positive Control

    PTEN and SMAD7 were the direct target genes of miR-216a. (A) In HCF cells, the relative luciferase activity was found to be significantly decreased when pGL3-PTEN-3’-UTR or pGL3-SMAD7-3’-UTR was transfected together with miR-216a mimic but not with the miRNA mimic control; (B) 72 hrs after the transfection in HCF cells, western blot revealed that the protein expression levels of PTEN and SMAD7 were significantly down-regulated in cells transfected with miR-216a mimic relative to those transfected with miRNA mimic control. *P

    Journal: Cardiovascular Diagnosis and Therapy

    Article Title: MiR-216a accelerates proliferation and fibrogenesis via targeting PTEN and SMAD7 in human cardiac fibroblasts

    doi: 10.21037/cdt.2019.11.06

    Figure Lengend Snippet: PTEN and SMAD7 were the direct target genes of miR-216a. (A) In HCF cells, the relative luciferase activity was found to be significantly decreased when pGL3-PTEN-3’-UTR or pGL3-SMAD7-3’-UTR was transfected together with miR-216a mimic but not with the miRNA mimic control; (B) 72 hrs after the transfection in HCF cells, western blot revealed that the protein expression levels of PTEN and SMAD7 were significantly down-regulated in cells transfected with miR-216a mimic relative to those transfected with miRNA mimic control. *P

    Article Snippet: The 3’UTRs of human PTEN and SMAD7 cDNA containing the putative target site for the miR-216a (sequence shown in Supplementary data) were chemically synthesized and inserted at the XbaI site, immediately downstream of the luciferase gene in the pGL3-control vector (Promega, Madison, WI, USA) by Integrated Biotech Solutions Co., Ltd (Shanghai, China), respectively.

    Techniques: Luciferase, Activity Assay, Transfection, Western Blot, Expressing

    Strategies for generation of dsRNAs and d-siRNAs. (A) PCR template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.

    Journal: PLoS ONE

    Article Title: Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans

    doi: 10.1371/journal.pone.0075443

    Figure Lengend Snippet: Strategies for generation of dsRNAs and d-siRNAs. (A) PCR template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.

    Article Snippet: The sGFP gene was PCR amplified from the pMT-sGFP vector and cloned in pGEM T-easy vector.

    Techniques: Polymerase Chain Reaction, Sequencing, Amplification, Plasmid Preparation, In Vitro, Polyacrylamide Gel Electrophoresis, Purification, Generated

    Generation and confirmation of Munc18-1-Venus (M18V) mouse. (A) Generation of M18V knockin gene. Diagrams indicate WT munc18-1 gene, targeting vector, M18V-neo-knock-in gene, and Cre-recombined M18V gene. Exons are indicated by gray boxes and numbered. Black and gray horizontal bars indicate probes used for Southern blot analysis and PCR products used for genotyping, respectively. A 5-kbp section is not shown (arrowheads). LoxP , loxP sites; Venus, Venus cDNA; AvrII , restriction enzyme site; NEO, neomycin resistance gene; TK, thymidine kinase promoter; pGEM-T Easy, targeting vector. (B) Southern blot analysis of mouse tail DNA from heterozygous (+/m) and WT (+/+) mice. DNA was AvrII-digested. m, M18V-neo-knock-in gene (8.2 kbp); +, munc18-1 gene (6.3 kbp). (C) Agarose gel of PCR products from +/+, +/m, and m/m mouse DNA. M18V, munc18-1-Venus gene PCR product, 449 bp; M18, munc18-1 gene PCR product, 195 bp. (D) Western blot of brain lysate of +/+, +/m, and m/m mice stained for M18V. Actin was used as a loading control. The lines indicate that intervening lanes have been spliced out. (E) Western blot of brain region lysate of m/m and +/+ mice at E18 stained for M18V. Valosin-containing protein (VCP) was used as a loading control. (F) Quantification of M18V expression levels in +/+ and m/m brain lysate. Homogenates from +/+ and m/m brains were analyzed by SDS-PAGE (10 µg of protein per lane) and Western blotting with Munc18-1 antibodies. M18V protein levels were normalized to VCP protein levels for each mouse ( n = 6). Error bars indicate mean ± SEM. (G) Hippocampal localization of M18V fluorescence in m/m (M18V homozygote) brain slice (left) and of antibody-stained Munc18-1 in WT (+/+) mice (right) compared with synaptic marker VAMP and dendritic marker MAP2. CA3, hippocampal CA region; Str, striatum; arrowheads, mossy fiber terminals of the stratum lucidum. Bars: (overview images) 100 µm; (enlarged panels) 25 µm.

    Journal: The Journal of Cell Biology

    Article Title: Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner

    doi: 10.1083/jcb.201308026

    Figure Lengend Snippet: Generation and confirmation of Munc18-1-Venus (M18V) mouse. (A) Generation of M18V knockin gene. Diagrams indicate WT munc18-1 gene, targeting vector, M18V-neo-knock-in gene, and Cre-recombined M18V gene. Exons are indicated by gray boxes and numbered. Black and gray horizontal bars indicate probes used for Southern blot analysis and PCR products used for genotyping, respectively. A 5-kbp section is not shown (arrowheads). LoxP , loxP sites; Venus, Venus cDNA; AvrII , restriction enzyme site; NEO, neomycin resistance gene; TK, thymidine kinase promoter; pGEM-T Easy, targeting vector. (B) Southern blot analysis of mouse tail DNA from heterozygous (+/m) and WT (+/+) mice. DNA was AvrII-digested. m, M18V-neo-knock-in gene (8.2 kbp); +, munc18-1 gene (6.3 kbp). (C) Agarose gel of PCR products from +/+, +/m, and m/m mouse DNA. M18V, munc18-1-Venus gene PCR product, 449 bp; M18, munc18-1 gene PCR product, 195 bp. (D) Western blot of brain lysate of +/+, +/m, and m/m mice stained for M18V. Actin was used as a loading control. The lines indicate that intervening lanes have been spliced out. (E) Western blot of brain region lysate of m/m and +/+ mice at E18 stained for M18V. Valosin-containing protein (VCP) was used as a loading control. (F) Quantification of M18V expression levels in +/+ and m/m brain lysate. Homogenates from +/+ and m/m brains were analyzed by SDS-PAGE (10 µg of protein per lane) and Western blotting with Munc18-1 antibodies. M18V protein levels were normalized to VCP protein levels for each mouse ( n = 6). Error bars indicate mean ± SEM. (G) Hippocampal localization of M18V fluorescence in m/m (M18V homozygote) brain slice (left) and of antibody-stained Munc18-1 in WT (+/+) mice (right) compared with synaptic marker VAMP and dendritic marker MAP2. CA3, hippocampal CA region; Str, striatum; arrowheads, mossy fiber terminals of the stratum lucidum. Bars: (overview images) 100 µm; (enlarged panels) 25 µm.

    Article Snippet: The left cloning arm, a genomic sequence 5′ of, and including, exon 20 of the munc18-1 gene from 129/Sv embryonic stem (ES) cell DNA, was subcloned using PCR into pGEM-T Easy (Promega), resulting in pGEM-T Easy-Left-Arm.

    Techniques: Knock-In, Plasmid Preparation, Southern Blot, Polymerase Chain Reaction, Mouse Assay, Agarose Gel Electrophoresis, Western Blot, Staining, Expressing, SDS Page, Fluorescence, Slice Preparation, Marker

    miR-144 regulates agrin expression by directly targeting the 3′ UTR of agrin mRNA. (A) Schematic representation of three putative miRNA-binding sites on the agrin 3′ UTR sequence. (B) The 3′ UTR fragment of agrin was cloned into the Xbal restriction site downstream of the firefly luciferase gene of the pGL3-Basic vector. Luciferase activity of each sample was measured 48h after transfection and normalized to Renilla luciferase activity. pGL3-Basic vector was used as control. Graph error bars indicate SD calculated from at least three independent experiments. * P

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Agrin Influences Botulinum Neurotoxin A-Induced Nerve Sprouting via miR-144-agrin-MuSK Signaling

    doi: 10.3389/fcell.2020.00015

    Figure Lengend Snippet: miR-144 regulates agrin expression by directly targeting the 3′ UTR of agrin mRNA. (A) Schematic representation of three putative miRNA-binding sites on the agrin 3′ UTR sequence. (B) The 3′ UTR fragment of agrin was cloned into the Xbal restriction site downstream of the firefly luciferase gene of the pGL3-Basic vector. Luciferase activity of each sample was measured 48h after transfection and normalized to Renilla luciferase activity. pGL3-Basic vector was used as control. Graph error bars indicate SD calculated from at least three independent experiments. * P

    Article Snippet: Luciferase Assay The 3′-UTR and mutant fragment of agrin and were subcloned into the Xbal restriction site downstream of the firefly luciferase gene of the pGL3-Basic Vector (Promega, United States) as pGL3-3′-UTR.

    Techniques: Expressing, Binding Assay, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Transfection

    miR-181a regulates the level of ETV6/RUNX1 . Overexpression of miR-181a in REH cells was performed by transfection with precursor miRNA. A final 50 nM concentration of nontargeting-miR (NC) or pre-mir-181a (181a) were transfected twice and cells were harvested after 48 hours of the second transfection for further examination. (A) miR-181a level was detected by TaqMan qRT-PCR. (B) Regulation of ETV6/RUNX1 by miR-181a was confirmed by Western blotting with RUNX1-specific (E/R) antibody. Relative expression as determined by densitometry is indicated below the blots. (C) The putative miR-181a binding site in the RUNX1 3' UTR. nt, nucleotides. (D) The last 678 bp of the human RUNX1 3' UTR containing normal (WT) or mutated (mut) miR-181a targeting sequences were cloned downstream of a pGL3-luciferase vector and transfected into 293FT cells with expression vectors for miR-181a (181a) or negative control shRNA (NC). All experiment was conducted in triplicate. Bars represent the mean ± SD of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (ANOVA).

    Journal: PLoS ONE

    Article Title: A Double Negative Loop Comprising ETV6/RUNX1 and MIR181A1 Contributes to Differentiation Block in t(12;21)-Positive Acute Lymphoblastic Leukemia

    doi: 10.1371/journal.pone.0142863

    Figure Lengend Snippet: miR-181a regulates the level of ETV6/RUNX1 . Overexpression of miR-181a in REH cells was performed by transfection with precursor miRNA. A final 50 nM concentration of nontargeting-miR (NC) or pre-mir-181a (181a) were transfected twice and cells were harvested after 48 hours of the second transfection for further examination. (A) miR-181a level was detected by TaqMan qRT-PCR. (B) Regulation of ETV6/RUNX1 by miR-181a was confirmed by Western blotting with RUNX1-specific (E/R) antibody. Relative expression as determined by densitometry is indicated below the blots. (C) The putative miR-181a binding site in the RUNX1 3' UTR. nt, nucleotides. (D) The last 678 bp of the human RUNX1 3' UTR containing normal (WT) or mutated (mut) miR-181a targeting sequences were cloned downstream of a pGL3-luciferase vector and transfected into 293FT cells with expression vectors for miR-181a (181a) or negative control shRNA (NC). All experiment was conducted in triplicate. Bars represent the mean ± SD of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (ANOVA).

    Article Snippet: A 678-bp fragment of the RUNX1 3′ UTR containing a binding site for miR-181a (UGAAUGU) was cloned into the XbaI site at the distal end of the luciferase reporter gene of pGL3-promoter vector (Promega).

    Techniques: Over Expression, Transfection, Concentration Assay, Quantitative RT-PCR, Western Blot, Expressing, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Negative Control, shRNA

    miR-214 post-transcriptionally regulates PTEN expression by targeting the 3’-UTR of PTEN. (A , B) Luciferase reporter assay were performed at 48 hr post-transfection. BGC-823 and SGC-7901 gastric cancer cell lines were transfected respectively with the Renilla luciferase expression construct pRL-TK and pGL3-PTEN-3’-UTR firefly luciferase expression construct, along with either anti-miR-214 or control anti-miRNA. Results showed that cells co-transfected with miR-214 and pGL3-PTEN plasmid exhibited a significant increase of reporter activity in comparison with those co-transfected with the control anti-miRNA and pGL3-PTEN plasmid. However, the reporter activity of cells co-transfected with anti-miR-214 and pGL3-PTEN-mut plasmid showed no significant difference with that of cells cotransfected with control microRNA and pGL3-PTEN-mut plasmid. (C , D) The expression level of PTEN protein was detected by Western Blot at 48 hr post-transfection and normalized to that of β-actin. Results showed that the level of PTEN protein was significantly increased in cells transfected with anti-miR-214 as compared to the cells transfected with control anti-miRNA. (E) The expression level of PTEN mRNA was detected by qRT-PCR at 48 hr posttransfection and normalized to that of GAPDH. Results showed that the expression level of PTEN mRNA exhibited no significantly difference between cells transfected with anti-miR-214 and those transfected with control anti-miRNA. Data represent mean ± SEM from three independent experiments; * P

    Journal: Cancer Cell International

    Article Title: MiR-214 regulate gastric cancer cell proliferation, migration and invasion by targeting PTEN

    doi: 10.1186/1475-2867-13-68

    Figure Lengend Snippet: miR-214 post-transcriptionally regulates PTEN expression by targeting the 3’-UTR of PTEN. (A , B) Luciferase reporter assay were performed at 48 hr post-transfection. BGC-823 and SGC-7901 gastric cancer cell lines were transfected respectively with the Renilla luciferase expression construct pRL-TK and pGL3-PTEN-3’-UTR firefly luciferase expression construct, along with either anti-miR-214 or control anti-miRNA. Results showed that cells co-transfected with miR-214 and pGL3-PTEN plasmid exhibited a significant increase of reporter activity in comparison with those co-transfected with the control anti-miRNA and pGL3-PTEN plasmid. However, the reporter activity of cells co-transfected with anti-miR-214 and pGL3-PTEN-mut plasmid showed no significant difference with that of cells cotransfected with control microRNA and pGL3-PTEN-mut plasmid. (C , D) The expression level of PTEN protein was detected by Western Blot at 48 hr post-transfection and normalized to that of β-actin. Results showed that the level of PTEN protein was significantly increased in cells transfected with anti-miR-214 as compared to the cells transfected with control anti-miRNA. (E) The expression level of PTEN mRNA was detected by qRT-PCR at 48 hr posttransfection and normalized to that of GAPDH. Results showed that the expression level of PTEN mRNA exhibited no significantly difference between cells transfected with anti-miR-214 and those transfected with control anti-miRNA. Data represent mean ± SEM from three independent experiments; * P

    Article Snippet: The full-length 3’-UTR segments of PTEN mRNA containing the miR-214 binding site was amplified by PCR and inserted into the Xba1-site of pGL3 vector (Promega, WI) and named pGL3-PTEN.

    Techniques: Expressing, Luciferase, Reporter Assay, Transfection, Construct, Plasmid Preparation, Activity Assay, Western Blot, Quantitative RT-PCR

    Reporter gene assay for the functional analysis of 3′-UTR regulated by miRNA. ( A ) The reporter construct contained the inserted DNA fragment of human SLC30A3 3′-UTR wild type (WT) or mutant (Mut) downstream of luc2 in the pmirGLO Dual-Luciferase miRNA Target Expression Vector. ( B ) Reporter gene assay was performed using CHO cells co-transfected with NC or miR-5572 mimic, and the reporter vector cloned with WT 3′-UTR of SLC30A3 or mutant 3′-UTR of SLC30A3 ( n = 3 in each experimental group). All data are presented as box and scatter plot. Statistical significance was determined by two-way ANOVA followed by post hoc Bonferroni’s test (* p

    Journal: International Journal of Molecular Sciences

    Article Title: MicroRNA-5572 Is a Novel MicroRNA-Regulating SLC30A3 in Sporadic Amyotrophic Lateral Sclerosis

    doi: 10.3390/ijms21124482

    Figure Lengend Snippet: Reporter gene assay for the functional analysis of 3′-UTR regulated by miRNA. ( A ) The reporter construct contained the inserted DNA fragment of human SLC30A3 3′-UTR wild type (WT) or mutant (Mut) downstream of luc2 in the pmirGLO Dual-Luciferase miRNA Target Expression Vector. ( B ) Reporter gene assay was performed using CHO cells co-transfected with NC or miR-5572 mimic, and the reporter vector cloned with WT 3′-UTR of SLC30A3 or mutant 3′-UTR of SLC30A3 ( n = 3 in each experimental group). All data are presented as box and scatter plot. Statistical significance was determined by two-way ANOVA followed by post hoc Bonferroni’s test (* p

    Article Snippet: The digested DNA fragment was then subcloned into the digested pmirGLO Dual-Luciferase miRNA Target Expression Vector.

    Techniques: Reporter Gene Assay, Functional Assay, Construct, Mutagenesis, Luciferase, Expressing, Plasmid Preparation, Transfection, Clone Assay

    miRNA-29c-3p directly binds in the 3′UTR of AKT3, TGIF2, CREB5, and CDK6 A . Target Scan predicted base pairing of mature miRNA-29c-3p seed sequences in the 3′UTRs of the indicated genes. B . Luciferase activity in MCF10.AT1 cells transfected with the dual luciferase vector pmiRGLo containing the wild type (Wt) and mutated (Mut) miRNA-29c-3p binding sites indicated in panel A. The cells were also transfected with an miRNA-29c-3p mimic or a negative control mimic. The firefly signal reported the gene regulation by miRNA binding and the internal control renilla luciferase was used for normalizing the transfection efficiency. * p

    Journal: Oncotarget

    Article Title: Regulation of miRNA-29c and its downstream pathways in preneoplastic progression of triple-negative breast cancer

    doi: 10.18632/oncotarget.14902

    Figure Lengend Snippet: miRNA-29c-3p directly binds in the 3′UTR of AKT3, TGIF2, CREB5, and CDK6 A . Target Scan predicted base pairing of mature miRNA-29c-3p seed sequences in the 3′UTRs of the indicated genes. B . Luciferase activity in MCF10.AT1 cells transfected with the dual luciferase vector pmiRGLo containing the wild type (Wt) and mutated (Mut) miRNA-29c-3p binding sites indicated in panel A. The cells were also transfected with an miRNA-29c-3p mimic or a negative control mimic. The firefly signal reported the gene regulation by miRNA binding and the internal control renilla luciferase was used for normalizing the transfection efficiency. * p

    Article Snippet: Cloning and luciferase assay The wild type and the 3- nucleotide mutated seed sequence of the broadly conserved binding sites of miRNA-29c-3p (as predicted by Target Scan) and 200 flanking nucleotides (both upstream and downstream) from the AKT3, CREB5, and TGIF2 of 3′UTR regions were PCR amplified from the MCF10.AT1 genomic DNA and cloned downstream of the firefly luciferase open reading frame at the PmeI and XbaI sites by using primers (described in ) in pmiRGLo vector (Promega Corporation, [Madison, WI]).

    Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Negative Control

    Pulsed field gel electrophoresis (PFGE) types for Xba I-digested genomic DNA of Salmonella enterica strains isolated from raw chicken meat Four different PFGE types ( A, B, C, D ) were generated according to serotypes. M – λ ladder marker for PFGE; ATCC – Salmonella Typhimurium ATCC 19585 control strain; LMM300 – S . Meleagridis; LMM288 – S . Heidelberg; LMM218 – S . Enteritidis; LMM182 – S . Heidelberg; LMM179 – S . Heidelberg; LMM175 – S . Heidelberg; LMM170 – S . Enteritidis; LMM120 – S . Typhimurium; LMM105 – S . Heidelberg.

    Journal: Germs

    Article Title: Molecular typing, antibiotic resistance profiles and biocide susceptibility in Salmonella enterica serotypes isolated from raw chicken meat marketed in Venezuela

    doi: 10.18683/germs.2019.1161

    Figure Lengend Snippet: Pulsed field gel electrophoresis (PFGE) types for Xba I-digested genomic DNA of Salmonella enterica strains isolated from raw chicken meat Four different PFGE types ( A, B, C, D ) were generated according to serotypes. M – λ ladder marker for PFGE; ATCC – Salmonella Typhimurium ATCC 19585 control strain; LMM300 – S . Meleagridis; LMM288 – S . Heidelberg; LMM218 – S . Enteritidis; LMM182 – S . Heidelberg; LMM179 – S . Heidelberg; LMM175 – S . Heidelberg; LMM170 – S . Enteritidis; LMM120 – S . Typhimurium; LMM105 – S . Heidelberg.

    Article Snippet: Slices of the prepared PFGE plugs were incubated with Xba I (Promega, Madison, WI, USA) at a concentration of 20 U/plug for 3 h at 37°C.

    Techniques: Pulsed-Field Gel, Electrophoresis, Isolation, Generated, Marker

    (A) Southern blot hybridization with the p1-9 probe of XbaI-digested genomic DNAs of S. enterica serovar Typhimurium DT104 control strain BN9181 carrying SGI1 (lane 2), serovar Virchow control strain SV20 carrying SGI1-J4 (lane 3), serovar Kentucky strain

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Early Strains of Multidrug-Resistant Salmonella enterica Serovar Kentucky Sequence Type 198 from Southeast Asia Harbor Salmonella Genomic Island 1-J Variants with a Novel Insertion Sequence

    doi: 10.1128/AAC.00732-12

    Figure Lengend Snippet: (A) Southern blot hybridization with the p1-9 probe of XbaI-digested genomic DNAs of S. enterica serovar Typhimurium DT104 control strain BN9181 carrying SGI1 (lane 2), serovar Virchow control strain SV20 carrying SGI1-J4 (lane 3), serovar Kentucky strain

    Article Snippet: The atypical insertion site of the complex integrons InSGI1-J (in ORF S023) was further assessed by Southern blot hybridization of whole genomic DNA cut by XbaI (Promega, Charbonnières, France) by using probe p1-9 as previously described ( , , , ).

    Techniques: Southern Blot, Hybridization

    Cell cycle-regulated expression of the CDC6 promoter is dependent on E2F DNA-binding sites. NIH 3T3 cells were transiently transfected with p[−130,+225], pGL3-DM, p[−570,+98], and p[−266,+98]. Forty-eight hours posttransfection, cells were serum starved for 24 h and subsequently stimulated with fresh medium containing 10% BCS. Lysates were made after the depicted time spans. Asynchronous (A) samples were valued as 100 adjusted luciferase counts, while the others were calculated in comparison to this. Transfection efficiencies were determined by pCMV-βgal cotransfection. Samples were obtained in duplicate, and the presented data are representative for at least three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Cell Cycle-Regulated Expression of Mammalian CDC6 Is Dependent on E2F

    doi:

    Figure Lengend Snippet: Cell cycle-regulated expression of the CDC6 promoter is dependent on E2F DNA-binding sites. NIH 3T3 cells were transiently transfected with p[−130,+225], pGL3-DM, p[−570,+98], and p[−266,+98]. Forty-eight hours posttransfection, cells were serum starved for 24 h and subsequently stimulated with fresh medium containing 10% BCS. Lysates were made after the depicted time spans. Asynchronous (A) samples were valued as 100 adjusted luciferase counts, while the others were calculated in comparison to this. Transfection efficiencies were determined by pCMV-βgal cotransfection. Samples were obtained in duplicate, and the presented data are representative for at least three independent experiments.

    Article Snippet: Mutated nucleotides are underlined. p[−1534,+225] was generated by cloning the 1.8-kb Sac I fragment from the Lambda Fix phage DNA into the Sac I site of pGL3-Basic. p[−570,+98] was generated by subcloning the internal Bam HI/ Bam HI fragment from p[−1534,+225] into the Bgl II site of pGL3-Basic. p[−266,+98] was generated by subcloning the internal Nae I/ Bam HI fragment into the Sma I and Bgl II sites of pGL3-Basic. pCMVHAhCDC6 was constructed by PCR amplification of the full-length open reading frame of hCDC6 , and the PCR product was subsequently cloned into the Bam HI site of pCMVHA ( ).

    Techniques: Expressing, Binding Assay, Transfection, Luciferase, Cotransfection

    EMSA using labeled wild-type (wt) and double mutant (DM) probes. CDC6 promoter probes were obtained by PCR on p[−130,+225] and pGL3-DM and used in combination with nuclear extract from MRC5 human fibroblasts. Specific E2F complexes that interact with the wt probe are depicted with a bracket to the right of the blot. Non-E2F containing protein-DNA complexes are given with asterisks to the left. The cold wt probe (in lane 3) and the cold mutant (derived from DM) were added in 50-fold excess over the quantity of radiolabeled fragment. Antibodies directed against DP-1, E2F-4, and pRB (lanes 5 to 9) shift different subsets of E2F-containing complexes, while the antibodies against p107 and p130 (lanes 10 to 12) do not. M1 (lane 13) is a monoclonal antibody raised against adenovirus E1A. FTβ (lane 14) is a polyclonal serum raised against farnesyl transferase. M1 and FTβ served as negative (neg.) controls. -, nothing added; PC, polyclonal antibody.

    Journal: Molecular and Cellular Biology

    Article Title: Cell Cycle-Regulated Expression of Mammalian CDC6 Is Dependent on E2F

    doi:

    Figure Lengend Snippet: EMSA using labeled wild-type (wt) and double mutant (DM) probes. CDC6 promoter probes were obtained by PCR on p[−130,+225] and pGL3-DM and used in combination with nuclear extract from MRC5 human fibroblasts. Specific E2F complexes that interact with the wt probe are depicted with a bracket to the right of the blot. Non-E2F containing protein-DNA complexes are given with asterisks to the left. The cold wt probe (in lane 3) and the cold mutant (derived from DM) were added in 50-fold excess over the quantity of radiolabeled fragment. Antibodies directed against DP-1, E2F-4, and pRB (lanes 5 to 9) shift different subsets of E2F-containing complexes, while the antibodies against p107 and p130 (lanes 10 to 12) do not. M1 (lane 13) is a monoclonal antibody raised against adenovirus E1A. FTβ (lane 14) is a polyclonal serum raised against farnesyl transferase. M1 and FTβ served as negative (neg.) controls. -, nothing added; PC, polyclonal antibody.

    Article Snippet: Mutated nucleotides are underlined. p[−1534,+225] was generated by cloning the 1.8-kb Sac I fragment from the Lambda Fix phage DNA into the Sac I site of pGL3-Basic. p[−570,+98] was generated by subcloning the internal Bam HI/ Bam HI fragment from p[−1534,+225] into the Bgl II site of pGL3-Basic. p[−266,+98] was generated by subcloning the internal Nae I/ Bam HI fragment into the Sma I and Bgl II sites of pGL3-Basic. pCMVHAhCDC6 was constructed by PCR amplification of the full-length open reading frame of hCDC6 , and the PCR product was subsequently cloned into the Bam HI site of pCMVHA ( ).

    Techniques: Labeling, Mutagenesis, Polymerase Chain Reaction, Derivative Assay

    Luciferase activity mediated by different CDC6 promoter constructs. (A) Schematic representation of the large human CDC6 promoter. The transcription start site (+1) is given by an arrow. Three putative E2F sites are depicted by black bars. The two E2F sites in the 355-bp fragment (shaded) are numbered 1 and 2. The endogenous restriction sites Bam HI and Nae I were used to construct p[−570,+98] and p[−266,+98]. Mutations introduced in the two most downstream E2F sites to obtain pGL3-DM, -SM1, and -SM2 are shown with big black crosses, and their sequences are given below the constructs. (B) Responses of p[−130,+225], pGL3-SM1, GL3-SM2, pGL3-DM, and p[−1534,+225] to E2F-1 and DP-1 cotransfection in U2-OS cells. The activity from the p[−130,+225] construct is upregulated approximately 13-fold by coexpression of E2F-1 and DP-1, while the mutant constructs pGL3-DM, -SM1, and -SM2 do respond significantly less to E2F-1 and DP-1. The larger p[−1534,+225] construct is upregulated approximately twofold. The p[−130,+225] construct without E2F-1 and/or DP-1 cotransfection is depicted as 100 adjusted luciferase counts after measuring β-Gal activity. All other values are given relative to this with standard deviations from the means.

    Journal: Molecular and Cellular Biology

    Article Title: Cell Cycle-Regulated Expression of Mammalian CDC6 Is Dependent on E2F

    doi:

    Figure Lengend Snippet: Luciferase activity mediated by different CDC6 promoter constructs. (A) Schematic representation of the large human CDC6 promoter. The transcription start site (+1) is given by an arrow. Three putative E2F sites are depicted by black bars. The two E2F sites in the 355-bp fragment (shaded) are numbered 1 and 2. The endogenous restriction sites Bam HI and Nae I were used to construct p[−570,+98] and p[−266,+98]. Mutations introduced in the two most downstream E2F sites to obtain pGL3-DM, -SM1, and -SM2 are shown with big black crosses, and their sequences are given below the constructs. (B) Responses of p[−130,+225], pGL3-SM1, GL3-SM2, pGL3-DM, and p[−1534,+225] to E2F-1 and DP-1 cotransfection in U2-OS cells. The activity from the p[−130,+225] construct is upregulated approximately 13-fold by coexpression of E2F-1 and DP-1, while the mutant constructs pGL3-DM, -SM1, and -SM2 do respond significantly less to E2F-1 and DP-1. The larger p[−1534,+225] construct is upregulated approximately twofold. The p[−130,+225] construct without E2F-1 and/or DP-1 cotransfection is depicted as 100 adjusted luciferase counts after measuring β-Gal activity. All other values are given relative to this with standard deviations from the means.

    Article Snippet: Mutated nucleotides are underlined. p[−1534,+225] was generated by cloning the 1.8-kb Sac I fragment from the Lambda Fix phage DNA into the Sac I site of pGL3-Basic. p[−570,+98] was generated by subcloning the internal Bam HI/ Bam HI fragment from p[−1534,+225] into the Bgl II site of pGL3-Basic. p[−266,+98] was generated by subcloning the internal Nae I/ Bam HI fragment into the Sma I and Bgl II sites of pGL3-Basic. pCMVHAhCDC6 was constructed by PCR amplification of the full-length open reading frame of hCDC6 , and the PCR product was subsequently cloned into the Bam HI site of pCMVHA ( ).

    Techniques: Luciferase, Activity Assay, Construct, Cotransfection, Mutagenesis

    Activity of the inhibitory element in transiently transfected cells. (A) Schematic diagram of constructs used in luciferase assays from transfected Vero cells highlighting the differences in the 5′ ends of these constructs. pGL3-MCS is a negative-control

    Journal:

    Article Title: Inhibition of Translation by a Short Element in the 5? Leader of the Herpes Simplex Virus 1 DNA Polymerase Transcript ▿

    doi: 10.1128/JVI.01484-07

    Figure Lengend Snippet: Activity of the inhibitory element in transiently transfected cells. (A) Schematic diagram of constructs used in luciferase assays from transfected Vero cells highlighting the differences in the 5′ ends of these constructs. pGL3-MCS is a negative-control

    Article Snippet: The firefly luciferase constructs used in transient-transfection assays were derived from pGL3-control, which drives expression of luciferase from the simian virus 40 (SV40) promoter. pGL3-MCS was created by ligating the Klenow-treated BssHII fragment containing the multiple cloning site (MCS) from pBS into pGL3-control that was digested with NcoI and HindIII and treated with Klenow and CIP. pGL3-pol5′UTR was created by ligating a Klenow-treated ClaI fragment containing the complete pol 5′ UTR from pBSpol-2-ClaI to pGL3-control that was digested with NcoI and HindIII and treated with Klenow and CIP. pGL3-pol-uORFmut was created in the same manner as pGL3-pol5′UTR, except that the ClaI fragment used in this case had the AUG codon of the uORF mutated to AAG by using QuikChange mutagenesis. pGL3-pol1-57 was generated by ligating a Klenow- and CIP-treated BamHI fragment from pGL3-pol5′UTR (this fragment contains the first 57 bases of the pol 5′ UTR but not the firefly luciferase ORF) to a Klenow-treated, NcoI- and BamHI-digested fragment from pGL3-control (this fragment contains the luciferase ORF). pGL3-pol58-208 was created by ligating a BamHI-digested, Klenow-treated, XbaI-digested fragment from pGL3-pol5′UTR (this fragment contains pol 5′ UTR bases 58 to 208 upstream of the firefly luciferase ORF) to pGL3-control that had been digested with HindIII, treated with Klenow, digested with XbaI, and treated with CIP.

    Techniques: Activity Assay, Transfection, Construct, Luciferase, Negative Control

    Inhibition of firefly luciferase activities by siRNAs targeted to TAR. ( A ) The inhibitory effects on pTAR-GL2, pGL3-3′TAR-FWD and pGL3-3′TAR-REV. Black bars show the remaining firefly luciferase activities when pTAR-GL2 was expressed in the presence of Tat protein. Gray and white bars show activities when pGL3-3′TAR-FWD (top) and pGL3-3′TAR-REV (bottom) were expressed in the presence and in the absence of Tat protein, respectively. ( B ) The inhibitory effects of 1, 10 and 100 nM siRNAs on pGL3-3′TAR-FWD (white bars) and pGL3-3′(Target site)-FWD (black bars) in the absence of Tat protein. ( C ) The inhibitory effects of siRNAs on pGL3-3′TAR-REV (white bars) and pGL3-3′(Target site)-REV (black bars) in the absence of Tat protein. ( D ) Annealing experiment for TAR1–TAR5 siRNAs using 5′ 32 P-labeled RNAs. Annealed siRNAs were separated from single-stranded RNA (ssRNA) by electrophoresis on a 20% native polyacrylamide gel.

    Journal: Nucleic Acids Research

    Article Title: Effects on RNAi of the tight structure, sequence and position of the targeted region

    doi: 10.1093/nar/gkh221

    Figure Lengend Snippet: Inhibition of firefly luciferase activities by siRNAs targeted to TAR. ( A ) The inhibitory effects on pTAR-GL2, pGL3-3′TAR-FWD and pGL3-3′TAR-REV. Black bars show the remaining firefly luciferase activities when pTAR-GL2 was expressed in the presence of Tat protein. Gray and white bars show activities when pGL3-3′TAR-FWD (top) and pGL3-3′TAR-REV (bottom) were expressed in the presence and in the absence of Tat protein, respectively. ( B ) The inhibitory effects of 1, 10 and 100 nM siRNAs on pGL3-3′TAR-FWD (white bars) and pGL3-3′(Target site)-FWD (black bars) in the absence of Tat protein. ( C ) The inhibitory effects of siRNAs on pGL3-3′TAR-REV (white bars) and pGL3-3′(Target site)-REV (black bars) in the absence of Tat protein. ( D ) Annealing experiment for TAR1–TAR5 siRNAs using 5′ 32 P-labeled RNAs. Annealed siRNAs were separated from single-stranded RNA (ssRNA) by electrophoresis on a 20% native polyacrylamide gel.

    Article Snippet: The amplified fragment was ligated into the XbaI site of pGL3, and two kinds of vector were obtained, one having the insert in the forward direction (pGL3-3′Stop-FWD) and one having the insert in the reverse direction (pGL3-3′Stop-REV).

    Techniques: Inhibition, Luciferase, Labeling, Electrophoresis

    Inhibition of firefly luciferase activities by siRNAs targeted to sites around the Luc2 site. ( A ) The inhibitory effects on pTAR-GL2 and pGL3. Black bars show the remaining firefly luciferase activities when pTAR-GL2 was expressed in the presence of Tat protein. Gray and white bars show luciferase activities when pGL3 was expressed in the presence and in the absence of Tat protein, respectively. An asterisk indicates that the transfected siRNA did not have a sequence that allowed it to target the reporter mRNA. ( B ) The inhibitory effects of siRNAs on pGL3-3′(Target site)-FWD (top) and pGL3-3′(Target site)-REV (bottom) in the absence of Tat protein. Note that each siRNA had two target sites in each mRNA because the original plasmid pGL3 had the same target sequence around the Luc2 site as that in pTAR-GL2.

    Journal: Nucleic Acids Research

    Article Title: Effects on RNAi of the tight structure, sequence and position of the targeted region

    doi: 10.1093/nar/gkh221

    Figure Lengend Snippet: Inhibition of firefly luciferase activities by siRNAs targeted to sites around the Luc2 site. ( A ) The inhibitory effects on pTAR-GL2 and pGL3. Black bars show the remaining firefly luciferase activities when pTAR-GL2 was expressed in the presence of Tat protein. Gray and white bars show luciferase activities when pGL3 was expressed in the presence and in the absence of Tat protein, respectively. An asterisk indicates that the transfected siRNA did not have a sequence that allowed it to target the reporter mRNA. ( B ) The inhibitory effects of siRNAs on pGL3-3′(Target site)-FWD (top) and pGL3-3′(Target site)-REV (bottom) in the absence of Tat protein. Note that each siRNA had two target sites in each mRNA because the original plasmid pGL3 had the same target sequence around the Luc2 site as that in pTAR-GL2.

    Article Snippet: The amplified fragment was ligated into the XbaI site of pGL3, and two kinds of vector were obtained, one having the insert in the forward direction (pGL3-3′Stop-FWD) and one having the insert in the reverse direction (pGL3-3′Stop-REV).

    Techniques: Inhibition, Luciferase, Transfection, Sequencing, Plasmid Preparation

    A codon-modified GAr sequence influences EBNA1 synthesis. ( A ) IVT assay of pcDNA3 expression constructs encoding EBNA1 (E1) (lane 1), E1ΔGA (lane 2), E1-GAr(100N) (lane 3), E1-GAr(100M) (lane 4), E1-GAr(200N) (lane 5), E1-GAr(200M) (lane 6), E1-GAr(300N) (lane 7), E1-GAr(300M) (lane 8), E1-GAr(400N) (lane 9), E1-GAr(400M) (lane 10), E1-GAr(500N) (lane 11), or E1-GAr(500M) (lane 12). The constructs were transcribed and translated in vitro with T7 RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. 35 S-methionine-labeled proteins were visualized by autoradiography. ( B and C ) Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities ( B ) or relative fold increase of EBNA1 encoded by codon-modified GAr domains compared with EBNA1 encoded by native GAr domains ( C ). ( D ) Western blot of EBV-negative HEK293 cells transfected with expression constructs encoding E1-GFP (lane 1), E1ΔGA-GFP (lane 2), E1-GAr(100N)-GFP (lane 3), E1-GAr(100M)-GFP (lane 4), E1-GAr(200N)-GFP (lane 5), E1-GAr(200M)-GFP (lane 6), E1-GAr(300N)-GFP (lane 7), E1-GAr(300M)-GFP (lane 8), E1-GAr(400N)-GFP (lane 9), E1-GAr(400M)-GFP (lane 10), E1-GAr(500N)-GFP (lane 11), or E1-GAr(500M)-GFP (lane 12) with a GFP antibody ( Upper ) or a monoclonal actin antibody ( Lower ). Molecular weight markers M r (kDa) are indicated on the left. ( E ) Band intensities after immunoblotting were quantified as described for B . Representative data from one of four experiments are presented here.

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

    Article Title: Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition

    doi: 10.1073/pnas.0801968105

    Figure Lengend Snippet: A codon-modified GAr sequence influences EBNA1 synthesis. ( A ) IVT assay of pcDNA3 expression constructs encoding EBNA1 (E1) (lane 1), E1ΔGA (lane 2), E1-GAr(100N) (lane 3), E1-GAr(100M) (lane 4), E1-GAr(200N) (lane 5), E1-GAr(200M) (lane 6), E1-GAr(300N) (lane 7), E1-GAr(300M) (lane 8), E1-GAr(400N) (lane 9), E1-GAr(400M) (lane 10), E1-GAr(500N) (lane 11), or E1-GAr(500M) (lane 12). The constructs were transcribed and translated in vitro with T7 RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. 35 S-methionine-labeled proteins were visualized by autoradiography. ( B and C ) Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities ( B ) or relative fold increase of EBNA1 encoded by codon-modified GAr domains compared with EBNA1 encoded by native GAr domains ( C ). ( D ) Western blot of EBV-negative HEK293 cells transfected with expression constructs encoding E1-GFP (lane 1), E1ΔGA-GFP (lane 2), E1-GAr(100N)-GFP (lane 3), E1-GAr(100M)-GFP (lane 4), E1-GAr(200N)-GFP (lane 5), E1-GAr(200M)-GFP (lane 6), E1-GAr(300N)-GFP (lane 7), E1-GAr(300M)-GFP (lane 8), E1-GAr(400N)-GFP (lane 9), E1-GAr(400M)-GFP (lane 10), E1-GAr(500N)-GFP (lane 11), or E1-GAr(500M)-GFP (lane 12) with a GFP antibody ( Upper ) or a monoclonal actin antibody ( Lower ). Molecular weight markers M r (kDa) are indicated on the left. ( E ) Band intensities after immunoblotting were quantified as described for B . Representative data from one of four experiments are presented here.

    Article Snippet: EBNA1/pcDNA3 expression constructs were linearized with XbaI and 1 μg of template transcribed with T7 RNA polymerase by using a Riboprobe in vitro transcription system (Promega) supplemented with 50 μCi [α-32 P]UTP (Amersham Biosciences).

    Techniques: Modification, Sequencing, Expressing, Construct, In Vitro, Labeling, Autoradiography, Software, Western Blot, Transfection, Molecular Weight

    Restriction maps of the pAX1 series vectors. The parental plasmid pAX1 is a derivative of pGEM-T (Table ), which contains a unique XbaI site that is flanked by DNA homologous to the intergenic region between PD702 and PD703. The DNA

    Journal: Applied and Environmental Microbiology

    Article Title: Chromosome-Based Genetic Complementation System for Xylella fastidiosa ▿

    doi: 10.1128/AEM.00024-09

    Figure Lengend Snippet: Restriction maps of the pAX1 series vectors. The parental plasmid pAX1 is a derivative of pGEM-T (Table ), which contains a unique XbaI site that is flanked by DNA homologous to the intergenic region between PD702 and PD703. The DNA

    Article Snippet: The resulting 1.6-kb fragment was then cloned into the pGEM-T vector (Promega), creating plasmid pAX1.

    Techniques: Plasmid Preparation

    Involvement of Ago2, TRBP2 and PACT in modified-siRNA-dependent gene silencing. ( A ) PACT and TRBP2 binding to siRNA (siLuc-36) and cognate siDNA. The 5′end of the guide strand of siRNA or siDNA was phosphorylated with [γ- 32 P] ATP and the 5′end of the passenger strand was phosphorylated with cold ATP. A mobility shift assay was carried out using PACT and TRBP2 proteins purified from E. coli cells. Note that TRBP2 and PACT are capable of binding to dsRNA but not dsDNA. Arrows indicate the position of protein–nucleic acid complexes. ( B ) Reduced expression of Ago2 and TRBP2 by RNAi. HeLa cells were transfected with siGY441 (control siRNA), siAgo2 or siTRBP2 (50 nM) along with pCAGIPuro-EGFP (0.5 µg) and subjected to RT–PCR 3 days after transfection. Note that Ago2 and TRBP2 RNA were specifically knocked down by siAgo2 and siTRBP2, respectively. ( C ) Requirement of Ago2 and TRBP2 for gene silencing due to transfection with modified siRNA with 8-bp GS-DNA substitution. HeLa cells were simultaneously transfected with pGL3-Control DNA (1 µg) and pRL-SV40 DNA (0.1 µg), and a mixture of siRNA and modified siRNA with DNA substitution. Two siRNAs, siLuc-36 (left panel) and siLuc-774 (right panel), and their modified siRNA counterparts, 5 nM each, were used for luc gene silencing. chiLuc-36 and chiLuc-774-GS are functional DNA-modified siRNAs specific to luc knockdown, whereas chiLuc-774-PS is a modified siRNA with DNA replacement in the PS-end-proximal region. Ago2 and TRBP2 were knocked down using 50 nM siAgo2 and siTRBP2, respectively. As an siRNA concentration control, siGY441 (50 nM), an siRNA for EGFP knockdown, was used. Gene-silencing activity was measured 24 h after transfection. Note both nonmodified and DNA-modified siRNA-dependent luc gene-silencing activity to be reduced by 30–50 points by knocking down Ago2 and TRBP2 activity through siAgo2 and siTRBP2 RNAi, respectively. ( D ) Functional DNA-modified siRNA-dependent target mRNA cleavage. HeLa cells were cotransfected with pTREC-2-153 and siLuc-1085 (a negative control siRNA), siLuc2-153 (target-cleavable siRNA) or its cognate modified siRNA with 8-bp GS-DNA substitution, each 5 nM. RNA was extracted 24 h after transfection, and cleavage sites were determined by primer extension. Sequence ladder was prepared using the same pTREC construct ( 25 ). Note that the position of the main cleavage site is precisely identical between RNAi and gene silencing due to transfection with modified siRNA with 8-bp GS-DNA substitution.

    Journal: Nucleic Acids Research

    Article Title: Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect

    doi: 10.1093/nar/gkn042

    Figure Lengend Snippet: Involvement of Ago2, TRBP2 and PACT in modified-siRNA-dependent gene silencing. ( A ) PACT and TRBP2 binding to siRNA (siLuc-36) and cognate siDNA. The 5′end of the guide strand of siRNA or siDNA was phosphorylated with [γ- 32 P] ATP and the 5′end of the passenger strand was phosphorylated with cold ATP. A mobility shift assay was carried out using PACT and TRBP2 proteins purified from E. coli cells. Note that TRBP2 and PACT are capable of binding to dsRNA but not dsDNA. Arrows indicate the position of protein–nucleic acid complexes. ( B ) Reduced expression of Ago2 and TRBP2 by RNAi. HeLa cells were transfected with siGY441 (control siRNA), siAgo2 or siTRBP2 (50 nM) along with pCAGIPuro-EGFP (0.5 µg) and subjected to RT–PCR 3 days after transfection. Note that Ago2 and TRBP2 RNA were specifically knocked down by siAgo2 and siTRBP2, respectively. ( C ) Requirement of Ago2 and TRBP2 for gene silencing due to transfection with modified siRNA with 8-bp GS-DNA substitution. HeLa cells were simultaneously transfected with pGL3-Control DNA (1 µg) and pRL-SV40 DNA (0.1 µg), and a mixture of siRNA and modified siRNA with DNA substitution. Two siRNAs, siLuc-36 (left panel) and siLuc-774 (right panel), and their modified siRNA counterparts, 5 nM each, were used for luc gene silencing. chiLuc-36 and chiLuc-774-GS are functional DNA-modified siRNAs specific to luc knockdown, whereas chiLuc-774-PS is a modified siRNA with DNA replacement in the PS-end-proximal region. Ago2 and TRBP2 were knocked down using 50 nM siAgo2 and siTRBP2, respectively. As an siRNA concentration control, siGY441 (50 nM), an siRNA for EGFP knockdown, was used. Gene-silencing activity was measured 24 h after transfection. Note both nonmodified and DNA-modified siRNA-dependent luc gene-silencing activity to be reduced by 30–50 points by knocking down Ago2 and TRBP2 activity through siAgo2 and siTRBP2 RNAi, respectively. ( D ) Functional DNA-modified siRNA-dependent target mRNA cleavage. HeLa cells were cotransfected with pTREC-2-153 and siLuc-1085 (a negative control siRNA), siLuc2-153 (target-cleavable siRNA) or its cognate modified siRNA with 8-bp GS-DNA substitution, each 5 nM. RNA was extracted 24 h after transfection, and cleavage sites were determined by primer extension. Sequence ladder was prepared using the same pTREC construct ( 25 ). Note that the position of the main cleavage site is precisely identical between RNAi and gene silencing due to transfection with modified siRNA with 8-bp GS-DNA substitution.

    Article Snippet: The assay for five siRNAs (siHPV16-497, 573, -698, -707 and -752) and cognate DNA-modified siRNAs (chiHPV16-497, 573, -698, -707 and -752) was carried out using pZeoSV2-hLuc. phLuc-Control is a derivative of pGL3-Control (Promega) in which the 1.7-kb XbaI/HindIII region encoding firefly luc is replaced with the XbaI/HindIII hLuc fragment of psiCHECK-2 (Promega). pZeoSV2-hLuc is constructed by introducing hLuc fragment into pZeoSV2 (Invitrogen).

    Techniques: Modification, Binding Assay, Mobility Shift, Purification, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Functional Assay, Concentration Assay, Activity Assay, Negative Control, Sequencing, Construct

    Comparison of seed-sequence-dependent off-target effects due to RNA and DNA seed arms. Off-target effects of 12 sets of functional siRNA and cognate modified siRNA with 8-bp GS-DNA substitution were examined using psiCHECK-sm and -cm plasmids with various sm and cm targets and the dual luciferase assay. ( A ) Structures of psiCHECK-sm and -cm plasmids and modified-siRNA-dependent mechanisms of sm and cm target recognition are schematically shown. Three thick green and orange arrows, respectively, indicate sm and cm target sequences inserted. Black thick horizontal bar, seed arm sequence (position 2–8) of the guide strand. DNA sequence in modified siRNA is colored in blue. Only the seed-sequence-including DNA portion of the guide strand possesses a complete complementarity to the sm target sequence which is situated in the 3′UTR of luc mRNA transcribed from psiCHECK-sm. The entire guide strand completely matches the cm sequence in luc mRNA transcribed from psiCHECK-cm. Luc, VIM and Oct, respectively, indicate to contain sequences related to firefly luc , human vimentin and mouse Oct4. ( B ) HeLa cells were transfected with psiCHECK-1 containing three repeats of sm or cm target sequences (0.1 µg) and either phLuc-Control or pGL3-Control (1 µg) was used as a control. Cells were simultaneously transfected with either nonmodified or DNA-modified siRNA at 50 n M. The lower graph shows silencing effects on cm targets, while the upper graph, those on sm targets. Results shown in the lower graph indicate that not only all nonmodified siRNAs but also modified siRNAs with 8-bp GS-DNA substitution are highly active in silencing cm target sequences, indicating that the guide strand in which an 8-bp region from the 5′ end is DNA is as active in gene silencing as that of cognate authentic siRNA. The upper graph shows that virtually no reduction in sm target expression can be seen in the case of transfection with modified siRNAs with 8-bp GS-DNA substitution. In contrast, occasional reduction in sm target expression occurred in the case of authentic siRNA treatment. These findings may indicate that gene silencing due to transfection of modified siRNAs with 8-bp GS-DNA substitution is associated less frequently, if any, with seed-sequence-dependent off-target effect than classical RNAi.

    Journal: Nucleic Acids Research

    Article Title: Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect

    doi: 10.1093/nar/gkn042

    Figure Lengend Snippet: Comparison of seed-sequence-dependent off-target effects due to RNA and DNA seed arms. Off-target effects of 12 sets of functional siRNA and cognate modified siRNA with 8-bp GS-DNA substitution were examined using psiCHECK-sm and -cm plasmids with various sm and cm targets and the dual luciferase assay. ( A ) Structures of psiCHECK-sm and -cm plasmids and modified-siRNA-dependent mechanisms of sm and cm target recognition are schematically shown. Three thick green and orange arrows, respectively, indicate sm and cm target sequences inserted. Black thick horizontal bar, seed arm sequence (position 2–8) of the guide strand. DNA sequence in modified siRNA is colored in blue. Only the seed-sequence-including DNA portion of the guide strand possesses a complete complementarity to the sm target sequence which is situated in the 3′UTR of luc mRNA transcribed from psiCHECK-sm. The entire guide strand completely matches the cm sequence in luc mRNA transcribed from psiCHECK-cm. Luc, VIM and Oct, respectively, indicate to contain sequences related to firefly luc , human vimentin and mouse Oct4. ( B ) HeLa cells were transfected with psiCHECK-1 containing three repeats of sm or cm target sequences (0.1 µg) and either phLuc-Control or pGL3-Control (1 µg) was used as a control. Cells were simultaneously transfected with either nonmodified or DNA-modified siRNA at 50 n M. The lower graph shows silencing effects on cm targets, while the upper graph, those on sm targets. Results shown in the lower graph indicate that not only all nonmodified siRNAs but also modified siRNAs with 8-bp GS-DNA substitution are highly active in silencing cm target sequences, indicating that the guide strand in which an 8-bp region from the 5′ end is DNA is as active in gene silencing as that of cognate authentic siRNA. The upper graph shows that virtually no reduction in sm target expression can be seen in the case of transfection with modified siRNAs with 8-bp GS-DNA substitution. In contrast, occasional reduction in sm target expression occurred in the case of authentic siRNA treatment. These findings may indicate that gene silencing due to transfection of modified siRNAs with 8-bp GS-DNA substitution is associated less frequently, if any, with seed-sequence-dependent off-target effect than classical RNAi.

    Article Snippet: The assay for five siRNAs (siHPV16-497, 573, -698, -707 and -752) and cognate DNA-modified siRNAs (chiHPV16-497, 573, -698, -707 and -752) was carried out using pZeoSV2-hLuc. phLuc-Control is a derivative of pGL3-Control (Promega) in which the 1.7-kb XbaI/HindIII region encoding firefly luc is replaced with the XbaI/HindIII hLuc fragment of psiCHECK-2 (Promega). pZeoSV2-hLuc is constructed by introducing hLuc fragment into pZeoSV2 (Invitrogen).

    Techniques: Sequencing, Functional Assay, Modification, Luciferase, Transfection, Expressing

    Effects of the size and position of deoxyribonucleotide substitutions on gene silencing. ( A ) siRNAs with various DNA substitutions were constructed based on the siLuc-36 sequence, which is shown in the lower margin with nucleotide position measured from the 5′ end of the guide strand, and were subjected to the luc gene assay. Six different types of DNA-modified siRNAs are schematically shown. In three left constructs, RNA sequences were progressively replaced with DNA counterparts from the PS end, which includes 5′ and 3′ ends of passenger and guide strands, respectively. In three right constructs, RNA was replaced with DNA from the GS end, including the 5 ′end of the guide strand and 3′ end of the passenger strand. Both left and right constructs include three types of DNA replacement, that is, double-strand replacement, and guide and passenger-specific replacement. DNA and RNA portions are colored in blue and orange, respectively, and arrows indicate the direction of DNA replacement. ( B–G ) Ribonucleotides of double (B, C), guide (D, E) and passenger (F, G) strands were progressively replaced with deoxyribonucleotide counterparts from the PS end (B, D, F) or the GS end (C, E, G). Numerals from -2 to 21, which are situated below each graph indicate nucleotide position from the 5′ end (position 1) of the guide strand. (–1, –2) and ( 20 , 21 ), respectively, correspond to 3′ overhangs at the GS and PS ends. Relative luc activity at position ‘x’ indicates the activity due to authentic siRNA (siLuc-36). S2, CHO-K1, HeLa and E14TG2a cells were transfected with pGL3-Control DNA (1 µg) and pRL-SV40 DNA (0.1 µg) with or without authentic siRNA or the corresponding modified siRNAs with various DNA substitution (50 nM each). Relative luc activity was measured 24 h after transfection. Note that modified siRNA with 10-bp or

    Journal: Nucleic Acids Research

    Article Title: Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect

    doi: 10.1093/nar/gkn042

    Figure Lengend Snippet: Effects of the size and position of deoxyribonucleotide substitutions on gene silencing. ( A ) siRNAs with various DNA substitutions were constructed based on the siLuc-36 sequence, which is shown in the lower margin with nucleotide position measured from the 5′ end of the guide strand, and were subjected to the luc gene assay. Six different types of DNA-modified siRNAs are schematically shown. In three left constructs, RNA sequences were progressively replaced with DNA counterparts from the PS end, which includes 5′ and 3′ ends of passenger and guide strands, respectively. In three right constructs, RNA was replaced with DNA from the GS end, including the 5 ′end of the guide strand and 3′ end of the passenger strand. Both left and right constructs include three types of DNA replacement, that is, double-strand replacement, and guide and passenger-specific replacement. DNA and RNA portions are colored in blue and orange, respectively, and arrows indicate the direction of DNA replacement. ( B–G ) Ribonucleotides of double (B, C), guide (D, E) and passenger (F, G) strands were progressively replaced with deoxyribonucleotide counterparts from the PS end (B, D, F) or the GS end (C, E, G). Numerals from -2 to 21, which are situated below each graph indicate nucleotide position from the 5′ end (position 1) of the guide strand. (–1, –2) and ( 20 , 21 ), respectively, correspond to 3′ overhangs at the GS and PS ends. Relative luc activity at position ‘x’ indicates the activity due to authentic siRNA (siLuc-36). S2, CHO-K1, HeLa and E14TG2a cells were transfected with pGL3-Control DNA (1 µg) and pRL-SV40 DNA (0.1 µg) with or without authentic siRNA or the corresponding modified siRNAs with various DNA substitution (50 nM each). Relative luc activity was measured 24 h after transfection. Note that modified siRNA with 10-bp or

    Article Snippet: The assay for five siRNAs (siHPV16-497, 573, -698, -707 and -752) and cognate DNA-modified siRNAs (chiHPV16-497, 573, -698, -707 and -752) was carried out using pZeoSV2-hLuc. phLuc-Control is a derivative of pGL3-Control (Promega) in which the 1.7-kb XbaI/HindIII region encoding firefly luc is replaced with the XbaI/HindIII hLuc fragment of psiCHECK-2 (Promega). pZeoSV2-hLuc is constructed by introducing hLuc fragment into pZeoSV2 (Invitrogen).

    Techniques: Construct, Sequencing, Gene Assay, Modification, Activity Assay, Transfection

    Gene expression efficacies of MENDs in BMDCs. ( a ) Schematic diagram of the R8-MEND (left) and KALA-MEND (right). ( b ) The R8-MEND and KALA-MEND encapsulating a conventional pDNA (pcDNA3.1-Luc; opened bar) or CpG-free pDNA (pCpGfree-Luc(0); closed bar) were transfected to BMDCs. Data were presented as the mean ± SD of three independent experiments. Statistical differences were evaluated by one-way ANOVA, followed by Student's t -test (** P

    Journal: Nucleic Acids Research

    Article Title: A KALA-modified lipid nanoparticle containing CpG-free plasmid DNA as a potential DNA vaccine carrier for antigen presentation and as an immune-stimulative adjuvant

    doi: 10.1093/nar/gkv008

    Figure Lengend Snippet: Gene expression efficacies of MENDs in BMDCs. ( a ) Schematic diagram of the R8-MEND (left) and KALA-MEND (right). ( b ) The R8-MEND and KALA-MEND encapsulating a conventional pDNA (pcDNA3.1-Luc; opened bar) or CpG-free pDNA (pCpGfree-Luc(0); closed bar) were transfected to BMDCs. Data were presented as the mean ± SD of three independent experiments. Statistical differences were evaluated by one-way ANOVA, followed by Student's t -test (** P

    Article Snippet: Plasmid construction Conventional pDNA encoding luciferase (GL3) was prepared by inserting a fragment encoding for GL3, obtained by the HindIII/XbaI digestion of the pGL3 basic vector (Promega, Madison, WI, USA) into the Hind III/XbaI-digested site of pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) ( ).

    Techniques: Expressing, Transfection

    MHC class-I restricted antigen presentation and in vivo CTL assay. ( a ) BMDCs were transfected with pcDNA3.1-OVA, pCpGfree-OVA(0) or pCpGfree-Luc(0) (KALA-MEND only) by means of R8-MEND or KALA-MEND. Transfected cells were co-cultured with a B3Z T-cell hybridoma for 15 h at 37°C. The co-cultured cells were then incubated with chlorophenol red β-D-galactopyranoside buffer for 4 h at 37°C. The absorbance at 595 nm was used as an index for antigen-presentation activity. The absorbance observed in non-treated BMDCs was subtracted from each group. Data are mean ± SD of three independent experiments. Non-detected (N.D.)means under the detection limit. Statistical analyses were performed by the one-way ANOVA, followed by Bonferroni test. ** P

    Journal: Nucleic Acids Research

    Article Title: A KALA-modified lipid nanoparticle containing CpG-free plasmid DNA as a potential DNA vaccine carrier for antigen presentation and as an immune-stimulative adjuvant

    doi: 10.1093/nar/gkv008

    Figure Lengend Snippet: MHC class-I restricted antigen presentation and in vivo CTL assay. ( a ) BMDCs were transfected with pcDNA3.1-OVA, pCpGfree-OVA(0) or pCpGfree-Luc(0) (KALA-MEND only) by means of R8-MEND or KALA-MEND. Transfected cells were co-cultured with a B3Z T-cell hybridoma for 15 h at 37°C. The co-cultured cells were then incubated with chlorophenol red β-D-galactopyranoside buffer for 4 h at 37°C. The absorbance at 595 nm was used as an index for antigen-presentation activity. The absorbance observed in non-treated BMDCs was subtracted from each group. Data are mean ± SD of three independent experiments. Non-detected (N.D.)means under the detection limit. Statistical analyses were performed by the one-way ANOVA, followed by Bonferroni test. ** P

    Article Snippet: Plasmid construction Conventional pDNA encoding luciferase (GL3) was prepared by inserting a fragment encoding for GL3, obtained by the HindIII/XbaI digestion of the pGL3 basic vector (Promega, Madison, WI, USA) into the Hind III/XbaI-digested site of pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) ( ).

    Techniques: In Vivo, CTL Assay, Transfection, Cell Culture, Incubation, Activity Assay

    miR-15b-5p, predicted to be induced in DFUs, and suppression of its targets, DNA repair and inflammatory regulatory genes. a. IPA identified miR-15b-5p as the top enriched miR predicted to target DFU regulated genes from microarrays. b. miR-15b-5p predicted targets comprise regulators of the inflammatory response and genes involved in DNA repair. c. IKBKB and WEE1, are down-regulated in HaCaT cells transfected with mimic miR-15b-5p. MSH2 and RAD50 showed a trend of down-regulation but did not reach statistical significance. ctrl = mimic negative control #1. d. IKBKB and WEE1 are down-regulated in S. aureus infected human ex-vivo wounds in comparison to control un-infected wounds. (n=3 independent experiments, paired t-test was used to determine statistical significance, * = p-value

    Journal: The Journal of investigative dermatology

    Article Title: STAPHYLOCOCCUS AUREUS TRIGGERS INDUCTION OF MIR-15B-5P TO DIMINISH DNA REPAIR AND DE-REGULATE INFLAMMATORY RESPONSE IN DIABETIC FOOT ULCERS

    doi: 10.1016/j.jid.2017.11.038

    Figure Lengend Snippet: miR-15b-5p, predicted to be induced in DFUs, and suppression of its targets, DNA repair and inflammatory regulatory genes. a. IPA identified miR-15b-5p as the top enriched miR predicted to target DFU regulated genes from microarrays. b. miR-15b-5p predicted targets comprise regulators of the inflammatory response and genes involved in DNA repair. c. IKBKB and WEE1, are down-regulated in HaCaT cells transfected with mimic miR-15b-5p. MSH2 and RAD50 showed a trend of down-regulation but did not reach statistical significance. ctrl = mimic negative control #1. d. IKBKB and WEE1 are down-regulated in S. aureus infected human ex-vivo wounds in comparison to control un-infected wounds. (n=3 independent experiments, paired t-test was used to determine statistical significance, * = p-value

    Article Snippet: The 3’UTRs of the WEE1 and IKBKB human genes were amplified by PCR from human genomic DNA with primers containing restriction sites for Sac I and Xba I ( ) and cloned into the pmirGLO Dual-Luciferase miRNA Target expression vector (Promega Corporation, Madison, WI).

    Techniques: Indirect Immunoperoxidase Assay, Transfection, Negative Control, Infection, Ex Vivo