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tiangen biotech co polymerase chain reaction genomic dna
Polymerase Chain Reaction Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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polymerase chain reaction genomic dna - by Bioz Stars, 2020-05
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DNA Extraction:

Article Title: Genetic analysis and molecular characterization of Chinese sesame (Sesamum indicum L.) cultivars using Insertion-Deletion (InDel) and Simple Sequence Repeat (SSR) markers
Article Snippet: .. Genomic DNA extraction and PCR Genomic DNA of 130 sesame accessions was all extracted from young leaves using the DNA extraction kit (TIANGEN Co. Ltd, Beijing). .. Polymerase chain reactions (PCR) for SSRs and InDels were performed in a 10 μl reactions, containing 10 ng DNA, 2 pmol of each primers, 2 nmol dNTPs, 15 nmol MgCl2 , 0.2 U Taq DNA polymerase (Fermentas, Canada) and 1X PCR buffer supplied together with the enzyme.

Article Title: Three novel mutations of the G6PC gene identified in Chinese patients with glycogen storage disease type Ia
Article Snippet: .. Genomic DNA isolation and polymerase chain reaction Genomic DNA of peripheral blood leucocytes was extracted routinely by isolation kit (Tiangen, China) according to the manufacturer’s instructions. .. All five coding exons and flanking introns of the G6PC gene were amplified by the use of primers listed in Table .

Article Title: Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China
Article Snippet: .. DNA Isolation and DNA Analysis with PCR Genomic DNA samples of patients (I 8, I 11, IV 1), 4 obligate carriers (I 2, II 1, III 1, III 4), and 100 healthy adults from our center were isolated from peripheral blood leukocytes using a DNA extraction kit (Tiangen, Beijing, China). .. The F9 exons were amplified in a total of 7 reactions using the primers and polymerase chain reaction (PCR) conditions shown in Table 1.

Article Title: Use of nested PCR for the detection of trichomonads in bronchoalveolar lavage fluid
Article Snippet: .. Genomic DNA extraction and polymerase chain reaction Genomic DNA was extracted using a TIANamp Blood DNA Kit (Tiangen, China) from 200 μl of BALF sediment. .. The 18S rRNA gene was first amplified using the primers TRC1-F (5′-GGTAATTCCAGCTCTGCG-3′) and TRC1-R (5′-TGGTAAGTTTCCCCGTGT-3′).

Article Title: Genetic mutations in non-syndromic deafness patients of uyghur and han chinese ethnicities in xinjiang, China: a comparative study
Article Snippet: .. Mutational analysis with PCR Genomic DNA was extracted from peripheral blood leukocytes using a commercial available DNA isolation kit (Tiangen Biotech Corporation, Beijing, China). .. The concentration and purity of DNA were determined using an ultraviolet spectrometry (ActGene Inc, Taipei, Taiwan).

Article Title: High-density genetic map construction and QTLs analysis of grain yield-related traits in Sesame (Sesamum indicum L.) based on RAD-Seq techonology
Article Snippet: .. Genomic DNA extraction and PCR Genomic DNA was extracted from young leaves using the DNA extraction kit (TIANGEN Co. Ltd, Beijing). .. One thousand two hundred and seventy-four PCR markers, including 134 genomic-SSRs, 1,061 EST-SSRs and 79 InDels were used for genetic map construction (Table ) [ ].

Polymerase Chain Reaction:

Article Title: Genetic analysis and molecular characterization of Chinese sesame (Sesamum indicum L.) cultivars using Insertion-Deletion (InDel) and Simple Sequence Repeat (SSR) markers
Article Snippet: .. Genomic DNA extraction and PCR Genomic DNA of 130 sesame accessions was all extracted from young leaves using the DNA extraction kit (TIANGEN Co. Ltd, Beijing). .. Polymerase chain reactions (PCR) for SSRs and InDels were performed in a 10 μl reactions, containing 10 ng DNA, 2 pmol of each primers, 2 nmol dNTPs, 15 nmol MgCl2 , 0.2 U Taq DNA polymerase (Fermentas, Canada) and 1X PCR buffer supplied together with the enzyme.

Article Title: Three novel mutations of the G6PC gene identified in Chinese patients with glycogen storage disease type Ia
Article Snippet: .. Genomic DNA isolation and polymerase chain reaction Genomic DNA of peripheral blood leucocytes was extracted routinely by isolation kit (Tiangen, China) according to the manufacturer’s instructions. .. All five coding exons and flanking introns of the G6PC gene were amplified by the use of primers listed in Table .

Article Title: Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China
Article Snippet: .. DNA Isolation and DNA Analysis with PCR Genomic DNA samples of patients (I 8, I 11, IV 1), 4 obligate carriers (I 2, II 1, III 1, III 4), and 100 healthy adults from our center were isolated from peripheral blood leukocytes using a DNA extraction kit (Tiangen, Beijing, China). .. The F9 exons were amplified in a total of 7 reactions using the primers and polymerase chain reaction (PCR) conditions shown in Table 1.

Article Title: Use of nested PCR for the detection of trichomonads in bronchoalveolar lavage fluid
Article Snippet: .. Genomic DNA extraction and polymerase chain reaction Genomic DNA was extracted using a TIANamp Blood DNA Kit (Tiangen, China) from 200 μl of BALF sediment. .. The 18S rRNA gene was first amplified using the primers TRC1-F (5′-GGTAATTCCAGCTCTGCG-3′) and TRC1-R (5′-TGGTAAGTTTCCCCGTGT-3′).

Article Title: Genetic mutations in non-syndromic deafness patients of uyghur and han chinese ethnicities in xinjiang, China: a comparative study
Article Snippet: .. Mutational analysis with PCR Genomic DNA was extracted from peripheral blood leukocytes using a commercial available DNA isolation kit (Tiangen Biotech Corporation, Beijing, China). .. The concentration and purity of DNA were determined using an ultraviolet spectrometry (ActGene Inc, Taipei, Taiwan).

Article Title: High-density genetic map construction and QTLs analysis of grain yield-related traits in Sesame (Sesamum indicum L.) based on RAD-Seq techonology
Article Snippet: .. Genomic DNA extraction and PCR Genomic DNA was extracted from young leaves using the DNA extraction kit (TIANGEN Co. Ltd, Beijing). .. One thousand two hundred and seventy-four PCR markers, including 134 genomic-SSRs, 1,061 EST-SSRs and 79 InDels were used for genetic map construction (Table ) [ ].

Isolation:

Article Title: Three novel mutations of the G6PC gene identified in Chinese patients with glycogen storage disease type Ia
Article Snippet: .. Genomic DNA isolation and polymerase chain reaction Genomic DNA of peripheral blood leucocytes was extracted routinely by isolation kit (Tiangen, China) according to the manufacturer’s instructions. .. All five coding exons and flanking introns of the G6PC gene were amplified by the use of primers listed in Table .

Article Title: Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China
Article Snippet: .. DNA Isolation and DNA Analysis with PCR Genomic DNA samples of patients (I 8, I 11, IV 1), 4 obligate carriers (I 2, II 1, III 1, III 4), and 100 healthy adults from our center were isolated from peripheral blood leukocytes using a DNA extraction kit (Tiangen, Beijing, China). .. The F9 exons were amplified in a total of 7 reactions using the primers and polymerase chain reaction (PCR) conditions shown in Table 1.

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    tiangen biotech co direct sequencing genomic dna
    Detection of BRAF gene mutation at exons 11 and 15. (A) Gel electrophoresis of the <t>PCR</t> products of exon 11 and 15 of the BRAF gene. The PCR product size of exon 11 is 356 bp, and that of exon 15 is 249 bp. Part of a sequence chromatogram from the Sanger sequencing of BRAF from (B) a patient and (C) the positive control. The red arrow indicates a BRAF mutation (T1799A) of exon 15 in the BCPAP papillary thyroid cancer cell line. BRAF, B-Raf proto-oncogene, serine/threonine kinase; PCR, polymerase chain reaction; M, <t>DNA</t> marker; NE, normal endometrium; EU, eutopic endometrium of endometriosis; EC, ectopic endometrium of endometriosis.
    Direct Sequencing Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/direct sequencing genomic dna/product/tiangen biotech co
    Average 91 stars, based on 1 article reviews
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    91
    tiangen biotech co dna sequencing genomic dna
    Disruption of CXCR4 in TZM-bl cells via lentiCRISPR/SaCas9 renders cells resistant to HIV-1 challenge. a CXCR4 gene disruption analysis in TZM-bl cells by the <t>T7E1</t> cleavage assay. Assays were performed as in Fig. 1 b. b Flow cytometry analysis of CXCR4 expression in lentivirus transduced TZM-bl cells. Assays were performed as in Fig. 1 c. c <t>DNA</t> sequences of CXCR4 of the transduced TZM-bl cells. Assays were performed as in Fig. 1 d. d Luciferase reporter assay to quantify HIV-1 infection level. Modified TZM-bl cells were infected with HIV-1 NL4-3 (MOI of 0.5 or 1) for 6 h, then washed three times with PBS and cultured in complete DMEM medium for 3 days. Cells were collected and lysed in 100 µl of lysis buffer (Promega) for luciferase assay. For ( b ), one representative out of three independent experiments is shown. For ( d ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P
    Dna Sequencing Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna sequencing genomic dna/product/tiangen biotech co
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna sequencing genomic dna - by Bioz Stars, 2020-05
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    93
    tiangen biotech co genomic dna
    Promoter methylation and histone H3 deacetylation were involved in <t>GPER</t> down regulation in CRC cell and tissues. The mRNA ( a ) and protein ( b ) levels of GPER in CRC cell lines, human colon mucosal epithelial NCM460 cells were measured by qRT-PCR and western blot analysis, respectively; ( c ) Methylation status of GPER promoter in CRC cell lines was determined by bisulfite genomic <t>DNA</t> sequencing. Each dot represents a CpG site. White dots represent unmethylated CpG dinucleotides whereas each black dots represents a methylated cytosine residue within the CpG islands; ( d ) Methylation statuses of GPER promoter in five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were determined by bisulfite genomic DNA sequencing; ( e ) HCT-116 or SW480 cells were treated with 5 μM 5-Aza for the different times, then mRNA of GPER was measured by use of qRT-PCR; ( f ) ChIP analysis of NCM460 and CRC cell lines were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody; ( g ) The correlation between relative acet-H3 enrichment and GPER mRNA expression in LS147T, HCT-116, SW620, HCT8, and SW480 cells; ( h ) ChIP analysis of five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody. Data were presented as means ± SD of three independent experiments. * p
    Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genomic dna/product/tiangen biotech co
    Average 93 stars, based on 11 article reviews
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    tiangen biotech co genomic dna isolation
    The effects of VgR knockout on the expression of Px VgR and Px Vg in P. xylostella . (A,D) The transcription level of PxVgR and PxVg genes were analyzed by <t>qRT-PCR,</t> however, (B,E) the expression patterns of Px VgR and Px Vg proteins were analyzed by Western blot. (C) The ovaries dissected from newly emerged MUT and WT females were successively treated with the Px VgR polyclonal antibody and Alex Fluor Plus 594-conjugated-secondary antibody (goat anti-rabbit) (red), and stained with DAPI Fluoromout-G TM for <t>DNA</t> (blue); bar = 50 um. The results were shown as mean ± SE. The asterisk ∗∗ above the bars represented significant difference ( P
    Genomic Dna Isolation, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Detection of BRAF gene mutation at exons 11 and 15. (A) Gel electrophoresis of the PCR products of exon 11 and 15 of the BRAF gene. The PCR product size of exon 11 is 356 bp, and that of exon 15 is 249 bp. Part of a sequence chromatogram from the Sanger sequencing of BRAF from (B) a patient and (C) the positive control. The red arrow indicates a BRAF mutation (T1799A) of exon 15 in the BCPAP papillary thyroid cancer cell line. BRAF, B-Raf proto-oncogene, serine/threonine kinase; PCR, polymerase chain reaction; M, DNA marker; NE, normal endometrium; EU, eutopic endometrium of endometriosis; EC, ectopic endometrium of endometriosis.

    Journal: International Journal of Molecular Medicine

    Article Title: Analysis of the oncogene BRAF mutation and the correlation of the expression of wild-type BRAF and CREB1 in endometriosis

    doi: 10.3892/ijmm.2017.3342

    Figure Lengend Snippet: Detection of BRAF gene mutation at exons 11 and 15. (A) Gel electrophoresis of the PCR products of exon 11 and 15 of the BRAF gene. The PCR product size of exon 11 is 356 bp, and that of exon 15 is 249 bp. Part of a sequence chromatogram from the Sanger sequencing of BRAF from (B) a patient and (C) the positive control. The red arrow indicates a BRAF mutation (T1799A) of exon 15 in the BCPAP papillary thyroid cancer cell line. BRAF, B-Raf proto-oncogene, serine/threonine kinase; PCR, polymerase chain reaction; M, DNA marker; NE, normal endometrium; EU, eutopic endometrium of endometriosis; EC, ectopic endometrium of endometriosis.

    Article Snippet: Genomic DNA isolation, PCR and direct sequencing Genomic DNA was extracted from freshly frozen tissue (100 mg) using the TIANamp Genomic DNA kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol.

    Techniques: Mutagenesis, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing, Positive Control, Marker

    Disruption of CXCR4 in TZM-bl cells via lentiCRISPR/SaCas9 renders cells resistant to HIV-1 challenge. a CXCR4 gene disruption analysis in TZM-bl cells by the T7E1 cleavage assay. Assays were performed as in Fig. 1 b. b Flow cytometry analysis of CXCR4 expression in lentivirus transduced TZM-bl cells. Assays were performed as in Fig. 1 c. c DNA sequences of CXCR4 of the transduced TZM-bl cells. Assays were performed as in Fig. 1 d. d Luciferase reporter assay to quantify HIV-1 infection level. Modified TZM-bl cells were infected with HIV-1 NL4-3 (MOI of 0.5 or 1) for 6 h, then washed three times with PBS and cultured in complete DMEM medium for 3 days. Cells were collected and lysed in 100 µl of lysis buffer (Promega) for luciferase assay. For ( b ), one representative out of three independent experiments is shown. For ( d ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P

    Journal: Retrovirology

    Article Title: Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection

    doi: 10.1186/s12977-017-0375-0

    Figure Lengend Snippet: Disruption of CXCR4 in TZM-bl cells via lentiCRISPR/SaCas9 renders cells resistant to HIV-1 challenge. a CXCR4 gene disruption analysis in TZM-bl cells by the T7E1 cleavage assay. Assays were performed as in Fig. 1 b. b Flow cytometry analysis of CXCR4 expression in lentivirus transduced TZM-bl cells. Assays were performed as in Fig. 1 c. c DNA sequences of CXCR4 of the transduced TZM-bl cells. Assays were performed as in Fig. 1 d. d Luciferase reporter assay to quantify HIV-1 infection level. Modified TZM-bl cells were infected with HIV-1 NL4-3 (MOI of 0.5 or 1) for 6 h, then washed three times with PBS and cultured in complete DMEM medium for 3 days. Cells were collected and lysed in 100 µl of lysis buffer (Promega) for luciferase assay. For ( b ), one representative out of three independent experiments is shown. For ( d ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P

    Article Snippet: T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing Genomic DNA was extracted with the Blood and Cell Culture DNA Midi kit (TianGen, China) according to the manufacturer’s instructions.

    Techniques: Cleavage Assay, Flow Cytometry, Cytometry, Expressing, Luciferase, Reporter Assay, Infection, Modification, Cell Culture, Lysis

    AAV-mediated SaCas9/sgRNAs delivery suppresses HIV-1 infection in human primary CD4 + T cells. a A schematic diagram of AAV transfer vectors containing SaCas9 endonuclease and sgRNA. b T7E1 cleavage assay after 5 days post-transduction. c DNA sequences of CXCR4 of the AAV transduced CD4 + T cells. d Flow cytometry analysis of CXCR4 expression in AAV transduced CD4 + T cells. Assays were performed as in Fig. 1 c. e CD4 + T cells counts at different times after transduction. CXCR4 disrupted cells continued to grow normally. f Flow cytometry analysis of apoptosis following AAV delivery SaCas9/sgRNA. g HIV-1 p24 was detected in the supernatants of the CD4 + T cells treated with AAV delivered SaCas9/sgRNA following HIV-1 NL4-3 infection. For ( d , e and f ), one representative out of three independent experiments is shown. For ( g ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P

    Journal: Retrovirology

    Article Title: Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection

    doi: 10.1186/s12977-017-0375-0

    Figure Lengend Snippet: AAV-mediated SaCas9/sgRNAs delivery suppresses HIV-1 infection in human primary CD4 + T cells. a A schematic diagram of AAV transfer vectors containing SaCas9 endonuclease and sgRNA. b T7E1 cleavage assay after 5 days post-transduction. c DNA sequences of CXCR4 of the AAV transduced CD4 + T cells. d Flow cytometry analysis of CXCR4 expression in AAV transduced CD4 + T cells. Assays were performed as in Fig. 1 c. e CD4 + T cells counts at different times after transduction. CXCR4 disrupted cells continued to grow normally. f Flow cytometry analysis of apoptosis following AAV delivery SaCas9/sgRNA. g HIV-1 p24 was detected in the supernatants of the CD4 + T cells treated with AAV delivered SaCas9/sgRNA following HIV-1 NL4-3 infection. For ( d , e and f ), one representative out of three independent experiments is shown. For ( g ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P

    Article Snippet: T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing Genomic DNA was extracted with the Blood and Cell Culture DNA Midi kit (TianGen, China) according to the manufacturer’s instructions.

    Techniques: Infection, Cleavage Assay, Transduction, Flow Cytometry, Cytometry, Expressing

    Genome editing of CXCR4 in Jurkat T cells confers cell inhibition to HIV-1 infection by lentiCRISPR/SaCas9. a CXCR4 gene disruption analysis in Jurkat T cells by T7E1 cleavage assay. Assays were performed as in Fig. 1 b. b Flow cytometry analysis of CXCR4 expression in lentivirus transduced Jurkat T cells. Assays were performed as in Fig. 1 c. c DNA sequences of CXCR4 of the transduced Jurkat T cells. Assays were performed as in Fig. 1 d. d HIV-1 p24 was detected in the Jurkat T cells treated with SaCas9/sgRNA. For ( b ), one representative out of three independent experiments is shown. For ( d ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t test (*** P

    Journal: Retrovirology

    Article Title: Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection

    doi: 10.1186/s12977-017-0375-0

    Figure Lengend Snippet: Genome editing of CXCR4 in Jurkat T cells confers cell inhibition to HIV-1 infection by lentiCRISPR/SaCas9. a CXCR4 gene disruption analysis in Jurkat T cells by T7E1 cleavage assay. Assays were performed as in Fig. 1 b. b Flow cytometry analysis of CXCR4 expression in lentivirus transduced Jurkat T cells. Assays were performed as in Fig. 1 c. c DNA sequences of CXCR4 of the transduced Jurkat T cells. Assays were performed as in Fig. 1 d. d HIV-1 p24 was detected in the Jurkat T cells treated with SaCas9/sgRNA. For ( b ), one representative out of three independent experiments is shown. For ( d ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t test (*** P

    Article Snippet: T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing Genomic DNA was extracted with the Blood and Cell Culture DNA Midi kit (TianGen, China) according to the manufacturer’s instructions.

    Techniques: Inhibition, Infection, Cleavage Assay, Flow Cytometry, Cytometry, Expressing

    CXCR4 gene silencing by lentivirus mediated CRISPR/SaCas9 delivery protects GHOST-X4 cells from HIV-1 infection. a A schematic diagram of lentiviral transfer vectors containing CRISPR/SaCas9 components. LentiCRISPR v2 plasmid was modified by replacing the SpCas9 with SaCas9. Then, based on the SaCas9 PAM sequence 5′-NNGRRT-3′, we designed, synthesized and cloned the CXCR4 sgRNAs into the vector using the Bsmb1 . b CXCR4 gene disruption analysis in GHOST-X4 cells by the T7E1 cleavage assay. GHOST-X4 cells were transduced with lentiviruses (MOI of 40) in the presence of polybrene for 12 h, and the genomic DNA was extracted and used as template to amplify a CXCR4 fragment (1100 bp). Con: lentiviral vectors expressing SaCas9 only, #8 and #9: lentiviral vectors expressing SaCas9/sgRNA #8 and #9. c Flow cytometry analysis of CXCR4 expression in lentivirus transduced GHOST-X4 cells. Neg, unstained cells. Con, #8 and #9 as samples described in ( b ) stained with anti-CXCR4-PE. d DNA sequences of CXCR4 of the transduced GHOST-X4 cells. PCR products were cloned into pGEM-T Easy vector and sequenced. The PAM sequences are lined and highlighted in red; the target sequences were shown in blue; deletions are indicated with (−) and insertions with (+). N/N indicates ratio of WT or mutations to total sequenced clones. e Flow cytometry analysis of transduced GHOST-X4 cells on GFP expression 3 days post HIV-1 NL4-3 infection. Neg, no HIV-1 infection. f HIV-1 NL4-3 infection was determined by p24 level. The cultured supernatants were collected at the indicated days post infection, and HIV-1 p24 level was determined using a p24 ELISA kit. For ( c and e ), one representative out of three independent experiments is shown. For ( f ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P

    Journal: Retrovirology

    Article Title: Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection

    doi: 10.1186/s12977-017-0375-0

    Figure Lengend Snippet: CXCR4 gene silencing by lentivirus mediated CRISPR/SaCas9 delivery protects GHOST-X4 cells from HIV-1 infection. a A schematic diagram of lentiviral transfer vectors containing CRISPR/SaCas9 components. LentiCRISPR v2 plasmid was modified by replacing the SpCas9 with SaCas9. Then, based on the SaCas9 PAM sequence 5′-NNGRRT-3′, we designed, synthesized and cloned the CXCR4 sgRNAs into the vector using the Bsmb1 . b CXCR4 gene disruption analysis in GHOST-X4 cells by the T7E1 cleavage assay. GHOST-X4 cells were transduced with lentiviruses (MOI of 40) in the presence of polybrene for 12 h, and the genomic DNA was extracted and used as template to amplify a CXCR4 fragment (1100 bp). Con: lentiviral vectors expressing SaCas9 only, #8 and #9: lentiviral vectors expressing SaCas9/sgRNA #8 and #9. c Flow cytometry analysis of CXCR4 expression in lentivirus transduced GHOST-X4 cells. Neg, unstained cells. Con, #8 and #9 as samples described in ( b ) stained with anti-CXCR4-PE. d DNA sequences of CXCR4 of the transduced GHOST-X4 cells. PCR products were cloned into pGEM-T Easy vector and sequenced. The PAM sequences are lined and highlighted in red; the target sequences were shown in blue; deletions are indicated with (−) and insertions with (+). N/N indicates ratio of WT or mutations to total sequenced clones. e Flow cytometry analysis of transduced GHOST-X4 cells on GFP expression 3 days post HIV-1 NL4-3 infection. Neg, no HIV-1 infection. f HIV-1 NL4-3 infection was determined by p24 level. The cultured supernatants were collected at the indicated days post infection, and HIV-1 p24 level was determined using a p24 ELISA kit. For ( c and e ), one representative out of three independent experiments is shown. For ( f ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P

    Article Snippet: T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing Genomic DNA was extracted with the Blood and Cell Culture DNA Midi kit (TianGen, China) according to the manufacturer’s instructions.

    Techniques: CRISPR, Infection, Plasmid Preparation, Modification, Sequencing, Synthesized, Clone Assay, Cleavage Assay, Transduction, Expressing, Flow Cytometry, Cytometry, Staining, Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    Promoter methylation and histone H3 deacetylation were involved in GPER down regulation in CRC cell and tissues. The mRNA ( a ) and protein ( b ) levels of GPER in CRC cell lines, human colon mucosal epithelial NCM460 cells were measured by qRT-PCR and western blot analysis, respectively; ( c ) Methylation status of GPER promoter in CRC cell lines was determined by bisulfite genomic DNA sequencing. Each dot represents a CpG site. White dots represent unmethylated CpG dinucleotides whereas each black dots represents a methylated cytosine residue within the CpG islands; ( d ) Methylation statuses of GPER promoter in five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were determined by bisulfite genomic DNA sequencing; ( e ) HCT-116 or SW480 cells were treated with 5 μM 5-Aza for the different times, then mRNA of GPER was measured by use of qRT-PCR; ( f ) ChIP analysis of NCM460 and CRC cell lines were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody; ( g ) The correlation between relative acet-H3 enrichment and GPER mRNA expression in LS147T, HCT-116, SW620, HCT8, and SW480 cells; ( h ) ChIP analysis of five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody. Data were presented as means ± SD of three independent experiments. * p

    Journal: Molecular Cancer

    Article Title: Epigenetic down regulation of G protein-coupled estrogen receptor (GPER) functions as a tumor suppressor in colorectal cancer

    doi: 10.1186/s12943-017-0654-3

    Figure Lengend Snippet: Promoter methylation and histone H3 deacetylation were involved in GPER down regulation in CRC cell and tissues. The mRNA ( a ) and protein ( b ) levels of GPER in CRC cell lines, human colon mucosal epithelial NCM460 cells were measured by qRT-PCR and western blot analysis, respectively; ( c ) Methylation status of GPER promoter in CRC cell lines was determined by bisulfite genomic DNA sequencing. Each dot represents a CpG site. White dots represent unmethylated CpG dinucleotides whereas each black dots represents a methylated cytosine residue within the CpG islands; ( d ) Methylation statuses of GPER promoter in five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were determined by bisulfite genomic DNA sequencing; ( e ) HCT-116 or SW480 cells were treated with 5 μM 5-Aza for the different times, then mRNA of GPER was measured by use of qRT-PCR; ( f ) ChIP analysis of NCM460 and CRC cell lines were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody; ( g ) The correlation between relative acet-H3 enrichment and GPER mRNA expression in LS147T, HCT-116, SW620, HCT8, and SW480 cells; ( h ) ChIP analysis of five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody. Data were presented as means ± SD of three independent experiments. * p

    Article Snippet: Bisulfite genomic DNA sequencing To analyze the methylation of GPER promoter, genomic DNA of CRC cells was prepared using TIANamp Genomic DNA kit (TIANGEN), followed by the treatment of sodium bisulfite using the Epitect Bisulfite DNA kit (QIAGEN, cat: 59824).

    Techniques: Methylation, Quantitative RT-PCR, Western Blot, DNA Sequencing, Chromatin Immunoprecipitation, Expressing

    The effects of VgR knockout on the expression of Px VgR and Px Vg in P. xylostella . (A,D) The transcription level of PxVgR and PxVg genes were analyzed by qRT-PCR, however, (B,E) the expression patterns of Px VgR and Px Vg proteins were analyzed by Western blot. (C) The ovaries dissected from newly emerged MUT and WT females were successively treated with the Px VgR polyclonal antibody and Alex Fluor Plus 594-conjugated-secondary antibody (goat anti-rabbit) (red), and stained with DAPI Fluoromout-G TM for DNA (blue); bar = 50 um. The results were shown as mean ± SE. The asterisk ∗∗ above the bars represented significant difference ( P

    Journal: Frontiers in Physiology

    Article Title: CRISPR/Cas9-Mediated Vitellogenin Receptor Knockout Leads to Functional Deficiency in the Reproductive Development of Plutella xylostella

    doi: 10.3389/fphys.2019.01585

    Figure Lengend Snippet: The effects of VgR knockout on the expression of Px VgR and Px Vg in P. xylostella . (A,D) The transcription level of PxVgR and PxVg genes were analyzed by qRT-PCR, however, (B,E) the expression patterns of Px VgR and Px Vg proteins were analyzed by Western blot. (C) The ovaries dissected from newly emerged MUT and WT females were successively treated with the Px VgR polyclonal antibody and Alex Fluor Plus 594-conjugated-secondary antibody (goat anti-rabbit) (red), and stained with DAPI Fluoromout-G TM for DNA (blue); bar = 50 um. The results were shown as mean ± SE. The asterisk ∗∗ above the bars represented significant difference ( P

    Article Snippet: Genomic DNA Isolation and PCR-Based Genotyping Genomic DNA (gDNA) was isolated from individual adults, using TIANamp Genomic DNA Kit (TIANGEN, China), following the manufacturer’s instructions.

    Techniques: Knock-Out, Expressing, Quantitative RT-PCR, Western Blot, Staining