genomic dna Tiangen Biotech Co Search Results


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  • 94
    tiangen biotech co genomic dna
    miR-29c-3p directly targets <t>DNA</t> methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p
    Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 8151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co tianamp genomic dna kit
    miR-29c-3p directly targets <t>DNA</t> methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p
    Tianamp Genomic Dna Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 4305 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co plant genomic dna kit
    Retention of <t>TYLCV</t> <t>DNA</t> by B. tabaci Q females after transfer to nonhost (cotton) plants as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h AAP on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on TYLCV-infected tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . (A) Relative quantity of TYLCV retained. (B) Rate at which TYLCV titer decreased in B. tabaci Q females. Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance; ∗ P
    Plant Genomic Dna Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 2007 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co genomic dna extraction kit
    The ChIP assay of S. cellulosum So ce <t>M4</t> containing recombinant TALE-VP64 vector: (A) the SDS-PAGE analysis of ChIP eluate; (B) the Western blot analysis of ChIP eluate; (C) the verification of the presence of P3 promoter in ChIP eluate: M. <t>DNA</t> marker, lanes 1–3 were positive control, blank control, and PCR product using ChIP eluate as a template.
    Genomic Dna Extraction Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 674 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co dna extraction kit
    The ChIP assay of S. cellulosum So ce <t>M4</t> containing recombinant TALE-VP64 vector: (A) the SDS-PAGE analysis of ChIP eluate; (B) the Western blot analysis of ChIP eluate; (C) the verification of the presence of P3 promoter in ChIP eluate: M. <t>DNA</t> marker, lanes 1–3 were positive control, blank control, and PCR product using ChIP eluate as a template.
    Dna Extraction Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 1992 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co genomic dna purification kit
    <t>DNA</t> sequencing analysis of the large DNA fragment (mutant hlyC , 3072-bp) amplified with hlyC flanking primers ( P11 , Table 1 ) from DNA of M . <t>hyorhinis</t> cells transformed with Mini-oriC-HT1 ( A ) and Mini-oriC-HT2 ( B ) targeting plasmids. The sequencing was performed in both forward and reverse directions using hlyC flanking primers, P11 (Table 1 )”
    Genomic Dna Purification Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co genomic dna isolation kit
    Growth of Hsp90 knockout parasite ( ΔHsp90 ) in vitro. The parasites were cultured in African green monkey kidney (Vero) cells, 10 5 T. gondii were added to the 6-well plates, and the infection ratio was 1:1. Observation of RH Δku80 (A), ΔHsp90 (B), and complemented (C) parasites by inverted microscope. The plaque produced by ΔPKAR strains (Fig. 6B) was significantly smaller than that of RH Δku80 and complemented parasites (Figs. 6A, 6C), scale bar = 20 μm. T. gondii tachyzoites and Vero cells were indicated by arrows, T. gondii (black arrow), Vero cells (white arrow). (D) The parasites were collected at the same time, and genomic <t>DNA</t> was extracted by <t>TIANGEN</t> kit. T. gondii DNA was detected by SYBR-green real-time PCR using B1 primer pairs, the standard curve was obtained by the known concentration of the RH Δku80 parasites with the primers (B1), and the parasite number was calculated by interpolation from this standard curve. ** p
    Genomic Dna Isolation Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co tianamp blood dna kit
    Growth of Hsp90 knockout parasite ( ΔHsp90 ) in vitro. The parasites were cultured in African green monkey kidney (Vero) cells, 10 5 T. gondii were added to the 6-well plates, and the infection ratio was 1:1. Observation of RH Δku80 (A), ΔHsp90 (B), and complemented (C) parasites by inverted microscope. The plaque produced by ΔPKAR strains (Fig. 6B) was significantly smaller than that of RH Δku80 and complemented parasites (Figs. 6A, 6C), scale bar = 20 μm. T. gondii tachyzoites and Vero cells were indicated by arrows, T. gondii (black arrow), Vero cells (white arrow). (D) The parasites were collected at the same time, and genomic <t>DNA</t> was extracted by <t>TIANGEN</t> kit. T. gondii DNA was detected by SYBR-green real-time PCR using B1 primer pairs, the standard curve was obtained by the known concentration of the RH Δku80 parasites with the primers (B1), and the parasite number was calculated by interpolation from this standard curve. ** p
    Tianamp Blood Dna Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 1905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co tianamp bacteria dna kit
    Growth of Hsp90 knockout parasite ( ΔHsp90 ) in vitro. The parasites were cultured in African green monkey kidney (Vero) cells, 10 5 T. gondii were added to the 6-well plates, and the infection ratio was 1:1. Observation of RH Δku80 (A), ΔHsp90 (B), and complemented (C) parasites by inverted microscope. The plaque produced by ΔPKAR strains (Fig. 6B) was significantly smaller than that of RH Δku80 and complemented parasites (Figs. 6A, 6C), scale bar = 20 μm. T. gondii tachyzoites and Vero cells were indicated by arrows, T. gondii (black arrow), Vero cells (white arrow). (D) The parasites were collected at the same time, and genomic <t>DNA</t> was extracted by <t>TIANGEN</t> kit. T. gondii DNA was detected by SYBR-green real-time PCR using B1 primer pairs, the standard curve was obtained by the known concentration of the RH Δku80 parasites with the primers (B1), and the parasite number was calculated by interpolation from this standard curve. ** p
    Tianamp Bacteria Dna Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 1974 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co genome dna
    Confirmation of ccpN point mutation and ccpN in-frame deletion by <t>PCR.</t> The PCR product was analyzed by agarose gel electrophoresis. A 1(Fermentas) was used as a molecular weight marker (lane M). A. Confirmation of the ccpN point mutation. Fragments were amplified using ccpN-Mut-P7/ccpN-Mut-P8 as primers. Each lane showed amplified <t>DNA</t> generated from a DNA template: lane 1, BUK-1C (Δ ccpN :: upp -cassette); lane 2, BUK-1 ( ccpN -mut-Ala 130 to Ser); lane 3, dsDNA PCR fragment (positive control); lane 4, BUK ( ccpN -wild type) (negative control). B. Confirmation of the ccpN in-frame deletion. Fragments were amplified using ccpN-Del-P1/ccpN-Del-P6 as primers. Each lane showed amplified DNA generated from a DNA template: lane 5, BUK-2C (Δ ccpN :: upp -cassette); lane 6, BUK-2 (Δ ccpN ); lane 7, dsDNA PCR fragment (positive control); lane 8, BUK ( ccpN -wild type) (negative control).
    Genome Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co dna extraction
    Confirmation of ccpN point mutation and ccpN in-frame deletion by <t>PCR.</t> The PCR product was analyzed by agarose gel electrophoresis. A 1(Fermentas) was used as a molecular weight marker (lane M). A. Confirmation of the ccpN point mutation. Fragments were amplified using ccpN-Mut-P7/ccpN-Mut-P8 as primers. Each lane showed amplified <t>DNA</t> generated from a DNA template: lane 1, BUK-1C (Δ ccpN :: upp -cassette); lane 2, BUK-1 ( ccpN -mut-Ala 130 to Ser); lane 3, dsDNA PCR fragment (positive control); lane 4, BUK ( ccpN -wild type) (negative control). B. Confirmation of the ccpN in-frame deletion. Fragments were amplified using ccpN-Del-P1/ccpN-Del-P6 as primers. Each lane showed amplified DNA generated from a DNA template: lane 5, BUK-2C (Δ ccpN :: upp -cassette); lane 6, BUK-2 (Δ ccpN ); lane 7, dsDNA PCR fragment (positive control); lane 8, BUK ( ccpN -wild type) (negative control).
    Dna Extraction, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co tianamp blood dna kit 0 1 1ml
    Confirmation of ccpN point mutation and ccpN in-frame deletion by <t>PCR.</t> The PCR product was analyzed by agarose gel electrophoresis. A 1(Fermentas) was used as a molecular weight marker (lane M). A. Confirmation of the ccpN point mutation. Fragments were amplified using ccpN-Mut-P7/ccpN-Mut-P8 as primers. Each lane showed amplified <t>DNA</t> generated from a DNA template: lane 1, BUK-1C (Δ ccpN :: upp -cassette); lane 2, BUK-1 ( ccpN -mut-Ala 130 to Ser); lane 3, dsDNA PCR fragment (positive control); lane 4, BUK ( ccpN -wild type) (negative control). B. Confirmation of the ccpN in-frame deletion. Fragments were amplified using ccpN-Del-P1/ccpN-Del-P6 as primers. Each lane showed amplified DNA generated from a DNA template: lane 5, BUK-2C (Δ ccpN :: upp -cassette); lane 6, BUK-2 (Δ ccpN ); lane 7, dsDNA PCR fragment (positive control); lane 8, BUK ( ccpN -wild type) (negative control).
    Tianamp Blood Dna Kit 0 1 1ml, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 90/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co bacterial genomic dna extraction kit
    Characterization of B. amyloliquefaciens strain <t>HAB-2</t> and its mutant. a Gel electrophoresis of PCR products of wild-type strain HAB-2 (lane HAB-2) and its mutant (lane Mutant); b MALDI-TOF MS analysis on the extract of HAB△ lpa mutant, 3000 bp <t>DNA</t> marker
    Bacterial Genomic Dna Extraction Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 90/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co dna isolation kit
    Characterization of B. amyloliquefaciens strain <t>HAB-2</t> and its mutant. a Gel electrophoresis of PCR products of wild-type strain HAB-2 (lane HAB-2) and its mutant (lane Mutant); b MALDI-TOF MS analysis on the extract of HAB△ lpa mutant, 3000 bp <t>DNA</t> marker
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    tiangen biotech co gdna dispelling rt supermix
    Characterization of B. amyloliquefaciens strain <t>HAB-2</t> and its mutant. a Gel electrophoresis of PCR products of wild-type strain HAB-2 (lane HAB-2) and its mutant (lane Mutant); b MALDI-TOF MS analysis on the extract of HAB△ lpa mutant, 3000 bp <t>DNA</t> marker
    Gdna Dispelling Rt Supermix, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co fastking gdna dispelling rt supermix
    Characterization of B. amyloliquefaciens strain <t>HAB-2</t> and its mutant. a Gel electrophoresis of PCR products of wild-type strain HAB-2 (lane HAB-2) and its mutant (lane Mutant); b MALDI-TOF MS analysis on the extract of HAB△ lpa mutant, 3000 bp <t>DNA</t> marker
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    miR-29c-3p directly targets DNA methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p

    Journal: Cell Death & Disease

    Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma

    doi: 10.1038/s41419-018-1281-7

    Figure Lengend Snippet: miR-29c-3p directly targets DNA methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p

    Article Snippet: Genomic DNA was isolated from HCC tumor tissues, paired normal adjacent tissues, and HCC cells using the DNA Isolation kit (Tiangen, Beijing, China) according to the manufacturer’s protocol.

    Techniques: Expressing

    DNA methyltransferase 3B (DNMT3B) is upregulated and large tumor suppressor gene 1 (LATS1) is downregulated in hepatocellular carcinoma (HCC). a Quantitative real-time PCR (qRT-PCR) analysis of DNMT3B expression in 150 pairs of HCC tissues and paired normal adjacent tissues. b qRT-PCR analysis of LATS1 expression in 150 pairs of HCC tissues and paired normal adjacent tissues. c Immunohistochemical staining analysis of DNMT3B protein expression levels in HCC tissues. d Immunohistochemical staining analysis of LATS1 protein expression levels in HCC tissues. e Kaplan–Meier analysis of overall survival between HCC patients with high and low DNMT3B expression. f Kaplan–Meier analysis of overall survival between high and low LATS1 expression in HCC patients. g Kaplan–Meier analysis of overall survival between the high miR-29c-3p/low DNMT3B/high LATS1 expression group and low miR-29c-3p/high DNMT3B/low LATS1 expression group; ** p

    Journal: Cell Death & Disease

    Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma

    doi: 10.1038/s41419-018-1281-7

    Figure Lengend Snippet: DNA methyltransferase 3B (DNMT3B) is upregulated and large tumor suppressor gene 1 (LATS1) is downregulated in hepatocellular carcinoma (HCC). a Quantitative real-time PCR (qRT-PCR) analysis of DNMT3B expression in 150 pairs of HCC tissues and paired normal adjacent tissues. b qRT-PCR analysis of LATS1 expression in 150 pairs of HCC tissues and paired normal adjacent tissues. c Immunohistochemical staining analysis of DNMT3B protein expression levels in HCC tissues. d Immunohistochemical staining analysis of LATS1 protein expression levels in HCC tissues. e Kaplan–Meier analysis of overall survival between HCC patients with high and low DNMT3B expression. f Kaplan–Meier analysis of overall survival between high and low LATS1 expression in HCC patients. g Kaplan–Meier analysis of overall survival between the high miR-29c-3p/low DNMT3B/high LATS1 expression group and low miR-29c-3p/high DNMT3B/low LATS1 expression group; ** p

    Article Snippet: Genomic DNA was isolated from HCC tumor tissues, paired normal adjacent tissues, and HCC cells using the DNA Isolation kit (Tiangen, Beijing, China) according to the manufacturer’s protocol.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Immunohistochemistry, Staining

    Rescue experiments are performed to confirm that DNA methyltransferase 3B (DNMT3B) is the functional target of miR-29c-3p in hepatocellular carcinoma (HCC) progression. a Western blot revealed DNMT3B protein expression in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p that were transfected with DNMT3B vector and NC. b Proliferation of MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and negative control (NC) was determined by CCK-8 assay. c Wound healing assay was performed to determine the effects of DNMT3B on HCC cell migration. d Colony formation assays assessed the effects of DNMT3B on HCC cell proliferation. e The methylation status of large tumor suppressor gene 1 (LATS1) was detected in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC. f Western blot revealed the expression of Hippo signaling pathway components, including proliferation- and apoptosis-related indicators in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC; * p

    Journal: Cell Death & Disease

    Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma

    doi: 10.1038/s41419-018-1281-7

    Figure Lengend Snippet: Rescue experiments are performed to confirm that DNA methyltransferase 3B (DNMT3B) is the functional target of miR-29c-3p in hepatocellular carcinoma (HCC) progression. a Western blot revealed DNMT3B protein expression in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p that were transfected with DNMT3B vector and NC. b Proliferation of MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and negative control (NC) was determined by CCK-8 assay. c Wound healing assay was performed to determine the effects of DNMT3B on HCC cell migration. d Colony formation assays assessed the effects of DNMT3B on HCC cell proliferation. e The methylation status of large tumor suppressor gene 1 (LATS1) was detected in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC. f Western blot revealed the expression of Hippo signaling pathway components, including proliferation- and apoptosis-related indicators in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC; * p

    Article Snippet: Genomic DNA was isolated from HCC tumor tissues, paired normal adjacent tissues, and HCC cells using the DNA Isolation kit (Tiangen, Beijing, China) according to the manufacturer’s protocol.

    Techniques: Functional Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, CCK-8 Assay, Wound Healing Assay, Migration, Methylation

    Aberrant DNA hypermethylation and expression of large tumor suppressor gene 1 (LATS1) in hepatocellular carcinoma (HCC) and HCC cell lines. a The methylation status of LATS1 was randomly detected in 7 HCC and paired normal adjacent tissues. b The methylation status of LATS1 was detected in LO2, MHCC-97H, HepG2, SMMC-7721, and Huh7cell lines. c Bisulfite sequencing analysis was performed on LATS1 promoter methylation in HCC tissues compared with paired normal adjacent tissues. d The relative mRNA expression of miR-29c-3p in 7 HCC and paired normal adjacent tissues. e The relative mRNA expression of DNA methyltransferase 3B (DNMT3B) in 7 HCC and paired normal adjacent tissues. f The relative mRNA expression of LATS1 in 7 HCC and paired normal adjacent tissues. g The CpG islands (shaded area) of LATS1 promoter region. h YAP and LATS1 protein expression in HCC and paired normal adjacent tissues. i YAP and LATS1 protein expression in HCC and LO2 cells; * p

    Journal: Cell Death & Disease

    Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma

    doi: 10.1038/s41419-018-1281-7

    Figure Lengend Snippet: Aberrant DNA hypermethylation and expression of large tumor suppressor gene 1 (LATS1) in hepatocellular carcinoma (HCC) and HCC cell lines. a The methylation status of LATS1 was randomly detected in 7 HCC and paired normal adjacent tissues. b The methylation status of LATS1 was detected in LO2, MHCC-97H, HepG2, SMMC-7721, and Huh7cell lines. c Bisulfite sequencing analysis was performed on LATS1 promoter methylation in HCC tissues compared with paired normal adjacent tissues. d The relative mRNA expression of miR-29c-3p in 7 HCC and paired normal adjacent tissues. e The relative mRNA expression of DNA methyltransferase 3B (DNMT3B) in 7 HCC and paired normal adjacent tissues. f The relative mRNA expression of LATS1 in 7 HCC and paired normal adjacent tissues. g The CpG islands (shaded area) of LATS1 promoter region. h YAP and LATS1 protein expression in HCC and paired normal adjacent tissues. i YAP and LATS1 protein expression in HCC and LO2 cells; * p

    Article Snippet: Genomic DNA was isolated from HCC tumor tissues, paired normal adjacent tissues, and HCC cells using the DNA Isolation kit (Tiangen, Beijing, China) according to the manufacturer’s protocol.

    Techniques: Expressing, Methylation, Methylation Sequencing

    The epigenetic regulator UHRF1 controls epigenetic silencing of RIP3 in cancer cells . ( a ) RKO cells, UHRF1-shRNA stable cell lines were generated as described in the Materials and Methods, and western blotting analysis of lysates from the indicated stable cell lines showing UHRF1, RIP3 and β -actin levels. RKO-UHRF1-shRNA: RKO cells stably expressing a shRNA targeting UHRF1. ( b ) Reverse transcription-PCR products from the indicate cells to detect the Rip3 mRNA. ( c ) Western blotting analysis of lysates from the indicated stable cell lines showing UHRF1, Flag, RIP3 and β -actin levels. Flag-UHRF1: UHRF1-shRNA cells transiently expressing an shRNA-resistant UHRF1. ( d ) RNAi of UHRF1 and Dnmt1 in LL/2 cells. Western blotting analysis of lysates from the indicated stable cell lines showing RIP3 and β -actin levels. ( e ) Methylation status of the CpG-dinucleotides of DNA sequences (−152 to +240 bp) upstream and downstream of RIP3 transcription start site was validated by bisulfite sequencing from the indicated cell lines. ( f ) RKO-UHRF1-shRNA cells were transfected with the control, RIP1 siRNA, RIP3 siRNA or MLKL siRNA. Forty-eight hours post transfection, cells were treated as indicated for additional 48 h. Cell viability was determined by measuring ATP levels. * P

    Journal: Cell Death & Disease

    Article Title: Regulation of RIP3 by the transcription factor Sp1 and the epigenetic regulator UHRF1 modulates cancer cell necroptosis

    doi: 10.1038/cddis.2017.483

    Figure Lengend Snippet: The epigenetic regulator UHRF1 controls epigenetic silencing of RIP3 in cancer cells . ( a ) RKO cells, UHRF1-shRNA stable cell lines were generated as described in the Materials and Methods, and western blotting analysis of lysates from the indicated stable cell lines showing UHRF1, RIP3 and β -actin levels. RKO-UHRF1-shRNA: RKO cells stably expressing a shRNA targeting UHRF1. ( b ) Reverse transcription-PCR products from the indicate cells to detect the Rip3 mRNA. ( c ) Western blotting analysis of lysates from the indicated stable cell lines showing UHRF1, Flag, RIP3 and β -actin levels. Flag-UHRF1: UHRF1-shRNA cells transiently expressing an shRNA-resistant UHRF1. ( d ) RNAi of UHRF1 and Dnmt1 in LL/2 cells. Western blotting analysis of lysates from the indicated stable cell lines showing RIP3 and β -actin levels. ( e ) Methylation status of the CpG-dinucleotides of DNA sequences (−152 to +240 bp) upstream and downstream of RIP3 transcription start site was validated by bisulfite sequencing from the indicated cell lines. ( f ) RKO-UHRF1-shRNA cells were transfected with the control, RIP1 siRNA, RIP3 siRNA or MLKL siRNA. Forty-eight hours post transfection, cells were treated as indicated for additional 48 h. Cell viability was determined by measuring ATP levels. * P

    Article Snippet: Genomic DNA extraction and bisulfite sequencing PCR Genomic DNA was extracted from cultured cells using TIANamp Genomic DNA Kit (Tiangen, Beijing, China), DP304.

    Techniques: shRNA, Stable Transfection, Generated, Western Blot, Expressing, Polymerase Chain Reaction, Methylation, Methylation Sequencing, Transfection

    Representative variation of DNA methylation pattern. H ( Eco R I + Hpa II digest) and M ( Eco R I + Msp I digest) refer to digestion with Eco R I + Hpa II and Eco R I + Msp I, respectively. “→” red arrows represent parts of differential methylated bands between common wheat and wheat carrying Gc chromosome(s). M stands for Marker DL2000. MA (1, 1), presence in both H ( Eco R I + Hpa II digest) and M ( Eco R I + Msp I digest) lanes; MB (1, 0), presence in H and absence in M lane; MC (0, 1), absence in H but presence in M lane; MD (0, 0) absence in both H and M lanes. CS: T. aestivum cv. Chinese Spring. CS–3C: monosomic addition line of Chinese Spring (CS) that carries a gametocidal chromosome 3C originated from Aegilops triuncialis . CS–3C3C: disomic addition line of Chinese Spring (CS) that carries two gametocidal chromosome 3C originated from Aegilops triuncialis .

    Journal: International Journal of Molecular Sciences

    Article Title: The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis

    doi: 10.3390/ijms18081738

    Figure Lengend Snippet: Representative variation of DNA methylation pattern. H ( Eco R I + Hpa II digest) and M ( Eco R I + Msp I digest) refer to digestion with Eco R I + Hpa II and Eco R I + Msp I, respectively. “→” red arrows represent parts of differential methylated bands between common wheat and wheat carrying Gc chromosome(s). M stands for Marker DL2000. MA (1, 1), presence in both H ( Eco R I + Hpa II digest) and M ( Eco R I + Msp I digest) lanes; MB (1, 0), presence in H and absence in M lane; MC (0, 1), absence in H but presence in M lane; MD (0, 0) absence in both H and M lanes. CS: T. aestivum cv. Chinese Spring. CS–3C: monosomic addition line of Chinese Spring (CS) that carries a gametocidal chromosome 3C originated from Aegilops triuncialis . CS–3C3C: disomic addition line of Chinese Spring (CS) that carries two gametocidal chromosome 3C originated from Aegilops triuncialis .

    Article Snippet: DNA Extraction Genomic DNA was extracted from anthers of CS, CS–3C and CS–3C3C using DNAsecure Plant Kit (TIANGEN, DP320, Beijing, China) according to the manufacturer’s instructions.

    Techniques: DNA Methylation Assay, Methylation, Marker

    The disruption rates induced by transfecting Cas9, eGFP and sgRNA co-expression vector for three genomic loci in different cancer cell lines. a and b show the disruption efficiency induced by transfecting Cas9, eGFP and sgRNA co-expression vector in c-ABL and BCR gene loci in K562 cells as determined by T7E1 assay. “−” represents cells without GFP sorting; “+” represents cells with GFP sorting; and “M” represents DNA size marker. c – e show the disruption rate induced by transfecting Cas9, eGFP and AAVS1-sgRNA co-expression vector in K562, MCF-7 and HCT-116 cells using TA cloning and sequencing analysis. Approximately 20–30 TA clones were randomly picked up for DNA sequencing, and the disruption rate (%) was calculated based on DNA sequencing. All cells were transfected with 4 μg of pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector. Seventy-two hours after transfection, GFP-positive cells were enriched through FACS sorting and genomic DNA was extracted from the sorted cells. The PCR amplification, T7E1 assay and TA cloning into PMD18-T vector were performed as described in “ Methods ”

    Journal: Cell & Bioscience

    Article Title: Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR–Cas9 and ssODN in human cancer cells

    doi: 10.1186/s13578-018-0200-z

    Figure Lengend Snippet: The disruption rates induced by transfecting Cas9, eGFP and sgRNA co-expression vector for three genomic loci in different cancer cell lines. a and b show the disruption efficiency induced by transfecting Cas9, eGFP and sgRNA co-expression vector in c-ABL and BCR gene loci in K562 cells as determined by T7E1 assay. “−” represents cells without GFP sorting; “+” represents cells with GFP sorting; and “M” represents DNA size marker. c – e show the disruption rate induced by transfecting Cas9, eGFP and AAVS1-sgRNA co-expression vector in K562, MCF-7 and HCT-116 cells using TA cloning and sequencing analysis. Approximately 20–30 TA clones were randomly picked up for DNA sequencing, and the disruption rate (%) was calculated based on DNA sequencing. All cells were transfected with 4 μg of pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector. Seventy-two hours after transfection, GFP-positive cells were enriched through FACS sorting and genomic DNA was extracted from the sorted cells. The PCR amplification, T7E1 assay and TA cloning into PMD18-T vector were performed as described in “ Methods ”

    Article Snippet: Genomic DNA isolation, PCR amplification and mutation detection Genomic DNA was extracted from harvested cells using a DNA isolation kit (Tiangen, Beijing, China).

    Techniques: Expressing, Plasmid Preparation, Marker, TA Cloning, Sequencing, Clone Assay, DNA Sequencing, Transfection, FACS, Polymerase Chain Reaction, Amplification

    Effect of ssODN homology length on insertion efficiency at the AAVS1 locus. K562 cells were nucleofected with 4 μg of pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector and 0.3 nmol of ssODN donors with different lengths. Cells were harvested 2-day post nucleofection, and the GFP-positive cells were sorted. Genomic DNA was isolated and 100 ng DNA was used for PCR amplification with a P2P4 primer pair ( a ), or P2P4 and F1R1 primer pairs ( b ). The numbers on the top of the gel images represent the homology length in nucleotides of a ssODN donor. A 20-mer donor has two 10-base homology arms. Each ssODN contains an Eco RI site between the homology arms. The DNA sequence of each ssODN is shown in Supplementary Table 1 . M: DNA Marker. NC: PCR control; CON1: normal cells; CON2: cells transfected with pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector only; CON3: cells transfected with ssODN donor (80 nucleotides) only; 20–80: cells transfected with pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector plus various lengths of ssODNA donors. Arrows indicate the predicted amplified fragments

    Journal: Cell & Bioscience

    Article Title: Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR–Cas9 and ssODN in human cancer cells

    doi: 10.1186/s13578-018-0200-z

    Figure Lengend Snippet: Effect of ssODN homology length on insertion efficiency at the AAVS1 locus. K562 cells were nucleofected with 4 μg of pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector and 0.3 nmol of ssODN donors with different lengths. Cells were harvested 2-day post nucleofection, and the GFP-positive cells were sorted. Genomic DNA was isolated and 100 ng DNA was used for PCR amplification with a P2P4 primer pair ( a ), or P2P4 and F1R1 primer pairs ( b ). The numbers on the top of the gel images represent the homology length in nucleotides of a ssODN donor. A 20-mer donor has two 10-base homology arms. Each ssODN contains an Eco RI site between the homology arms. The DNA sequence of each ssODN is shown in Supplementary Table 1 . M: DNA Marker. NC: PCR control; CON1: normal cells; CON2: cells transfected with pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector only; CON3: cells transfected with ssODN donor (80 nucleotides) only; 20–80: cells transfected with pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector plus various lengths of ssODNA donors. Arrows indicate the predicted amplified fragments

    Article Snippet: Genomic DNA isolation, PCR amplification and mutation detection Genomic DNA was extracted from harvested cells using a DNA isolation kit (Tiangen, Beijing, China).

    Techniques: Expressing, Plasmid Preparation, Isolation, Polymerase Chain Reaction, Amplification, Sequencing, Marker, Transfection

    Determination of insertion repair efficiency and NHEJ rate at AAVS1 locus by restriction fragment length polymorphism assay, TA cloning and DNA sequencing. a and b show the enhancement of insertion repair by ssODN and SCR7 treatment in MCF-7 and HCT-116 cells as determined by restriction fragment length polymorphism assay. Genomic DNA was amplified by PCR, digested with Eco RI, and resolved in a 10% acrylamide gel. The original and cleaved DNA fragments are marked by arrows; the signals were quantified by densitometry; and the percentages of the cleaved fragments were calculated as described in “ Methods ”. c and d show the quantitation of insertion repair efficiency (%HDR), and Panels e and f show the percentages of NHEJ at AAVS1 locus by DNA sequencing of TA clones in MCF-7 and HCT-116 cells, respectively. Approximately 100 TA-clones were randomly picked up for DNA sequencing, and the insertion repair efficiency (%HDR) and NHEJ rate (%) in MCF-7 and HCT-116 cells was calculated based on DNA sequencing as described in the Methods. The data are the mean ± SD of three independent experiments. *p

    Journal: Cell & Bioscience

    Article Title: Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR–Cas9 and ssODN in human cancer cells

    doi: 10.1186/s13578-018-0200-z

    Figure Lengend Snippet: Determination of insertion repair efficiency and NHEJ rate at AAVS1 locus by restriction fragment length polymorphism assay, TA cloning and DNA sequencing. a and b show the enhancement of insertion repair by ssODN and SCR7 treatment in MCF-7 and HCT-116 cells as determined by restriction fragment length polymorphism assay. Genomic DNA was amplified by PCR, digested with Eco RI, and resolved in a 10% acrylamide gel. The original and cleaved DNA fragments are marked by arrows; the signals were quantified by densitometry; and the percentages of the cleaved fragments were calculated as described in “ Methods ”. c and d show the quantitation of insertion repair efficiency (%HDR), and Panels e and f show the percentages of NHEJ at AAVS1 locus by DNA sequencing of TA clones in MCF-7 and HCT-116 cells, respectively. Approximately 100 TA-clones were randomly picked up for DNA sequencing, and the insertion repair efficiency (%HDR) and NHEJ rate (%) in MCF-7 and HCT-116 cells was calculated based on DNA sequencing as described in the Methods. The data are the mean ± SD of three independent experiments. *p

    Article Snippet: Genomic DNA isolation, PCR amplification and mutation detection Genomic DNA was extracted from harvested cells using a DNA isolation kit (Tiangen, Beijing, China).

    Techniques: Non-Homologous End Joining, Polymorphism Assay, TA Cloning, DNA Sequencing, Amplification, Polymerase Chain Reaction, Acrylamide Gel Assay, Quantitation Assay, Clone Assay

    SCR7 promoted insertion repair efficiency at AAVS1 locus. a schematically illustrates the insertion repair mediated by ssODN and CRISPR/Cas9 for AAVS1, and the three methods used for the detection of insertion repair efficiency. A pair of primer P2 and P4 were used to examine insertion repair occurred in the Cas9-targeted locus by semi-quantitative PCR-gel electrophoresis. The two pairs of primers F3 and R3, F1 and R1 were used to amplify the sequences involved in the targeted site. The PCR-amplified products were analyzed by RFLP assay following Eco RI digestion and by TA cloning and DNA sequencing. b and c show representative images of PCR amplification with P2 and P4 primer and gel electrophoresis from at least three independent experiments. The data shows the enhancement of insertion repair by ssODN and SCR7 treatment in MCF-7 and HCT-116 cells in a SCR7 dose-dependent manner. “M”—DNA markers, “BC”—blank control without cells, “Con”—control cells without transfection of ssODN and CRISPR/Cas9 vector. d and e show the quantitative data of PCR-gel electrophoresis analysis using Image J software. GAPDH was used as an internal control. The data is the mean ± SD of three independent experiments. *p

    Journal: Cell & Bioscience

    Article Title: Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR–Cas9 and ssODN in human cancer cells

    doi: 10.1186/s13578-018-0200-z

    Figure Lengend Snippet: SCR7 promoted insertion repair efficiency at AAVS1 locus. a schematically illustrates the insertion repair mediated by ssODN and CRISPR/Cas9 for AAVS1, and the three methods used for the detection of insertion repair efficiency. A pair of primer P2 and P4 were used to examine insertion repair occurred in the Cas9-targeted locus by semi-quantitative PCR-gel electrophoresis. The two pairs of primers F3 and R3, F1 and R1 were used to amplify the sequences involved in the targeted site. The PCR-amplified products were analyzed by RFLP assay following Eco RI digestion and by TA cloning and DNA sequencing. b and c show representative images of PCR amplification with P2 and P4 primer and gel electrophoresis from at least three independent experiments. The data shows the enhancement of insertion repair by ssODN and SCR7 treatment in MCF-7 and HCT-116 cells in a SCR7 dose-dependent manner. “M”—DNA markers, “BC”—blank control without cells, “Con”—control cells without transfection of ssODN and CRISPR/Cas9 vector. d and e show the quantitative data of PCR-gel electrophoresis analysis using Image J software. GAPDH was used as an internal control. The data is the mean ± SD of three independent experiments. *p

    Article Snippet: Genomic DNA isolation, PCR amplification and mutation detection Genomic DNA was extracted from harvested cells using a DNA isolation kit (Tiangen, Beijing, China).

    Techniques: CRISPR, Real-time Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, RFLP Assay, TA Cloning, DNA Sequencing, Transfection, Plasmid Preparation, Software

    Retention of TYLCV DNA by B. tabaci Q females after transfer to nonhost (cotton) plants as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h AAP on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on TYLCV-infected tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . (A) Relative quantity of TYLCV retained. (B) Rate at which TYLCV titer decreased in B. tabaci Q females. Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance; ∗ P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Retention of TYLCV DNA by B. tabaci Q females after transfer to nonhost (cotton) plants as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h AAP on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on TYLCV-infected tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . (A) Relative quantity of TYLCV retained. (B) Rate at which TYLCV titer decreased in B. tabaci Q females. Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance; ∗ P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques: Infection

    Acquisition of TYLCV DNA by B. tabaci Q females from tomato plants inoculated in different order with both TSWV and TYLCV. TSWV-TYLCV: plants were inoculated with TSWV and 2 weeks later with TYLCV. TYLCV-TSWV: plants were inoculated with TYLCV and 2 weeks later with TSWV. Concurrent: plants were simultaneously inoculated with both viruses. Control: plants were inoculated with TYLCV and were mock-inoculated 2 weeks later. (A) Acquisition ability of TYLCV DNA by B. tabaci Q females from TSWV-TYLCV plants. (B) Acquisition ability of TYLCV DNA by B. tabaci Q females from concurrent plants. (C) Acquisition ability of TYLCV DNA by B. tabaci Q females from TYLCV-TSWV plants. Values are means ± SE. Asterisks indicate significant differences between treatments vs. control (one-way analysis of variance; ∗ P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Acquisition of TYLCV DNA by B. tabaci Q females from tomato plants inoculated in different order with both TSWV and TYLCV. TSWV-TYLCV: plants were inoculated with TSWV and 2 weeks later with TYLCV. TYLCV-TSWV: plants were inoculated with TYLCV and 2 weeks later with TSWV. Concurrent: plants were simultaneously inoculated with both viruses. Control: plants were inoculated with TYLCV and were mock-inoculated 2 weeks later. (A) Acquisition ability of TYLCV DNA by B. tabaci Q females from TSWV-TYLCV plants. (B) Acquisition ability of TYLCV DNA by B. tabaci Q females from concurrent plants. (C) Acquisition ability of TYLCV DNA by B. tabaci Q females from TYLCV-TSWV plants. Values are means ± SE. Asterisks indicate significant differences between treatments vs. control (one-way analysis of variance; ∗ P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques:

    Transmission of TYLCV DNA to tomato plants by Bemisia tabaci Q females as affected by previous infection of the tomato plants by TSWV. TSWV + or TSWV - : Tomato plants inoculated with TSWV or mock-inoculated, respectively, before they were exposed to Bemisia tabaci Q females that contained TYLCV. TYLCV + : Females that acquired TYLCV from another TYLCV-infected plant. Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗∗ P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Transmission of TYLCV DNA to tomato plants by Bemisia tabaci Q females as affected by previous infection of the tomato plants by TSWV. TSWV + or TSWV - : Tomato plants inoculated with TSWV or mock-inoculated, respectively, before they were exposed to Bemisia tabaci Q females that contained TYLCV. TYLCV + : Females that acquired TYLCV from another TYLCV-infected plant. Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗∗ P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques: Transmission Assay, Infection

    Transmission of TYLCV DNA to tomato plants by Bemisia tabaci Q females as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h AAP on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗∗ P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Transmission of TYLCV DNA to tomato plants by Bemisia tabaci Q females as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h AAP on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗∗ P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques: Transmission Assay, Infection

    Acquisition of TYLCV by Frankliniella occidentalis (A) , fecundity of F. occidentalis as affected by TYLCV (B) , and transmission of TSWV by F. occidentalis as affected by previous exposure of the thrips to TYLCV (C) . (A) PCR amplification of TYLCV was performed with the DNA extracted from F. occidentalis second-instar nymphs (lanes 3–7; one nymph per lane) and newly emerged adults (lanes 8–12; one adult per lane) obtained from a TYLCV-infected tomato plant. Amplification products were visualized by agarose gel electrophoresis with Gelview staining. Lane 1: positive control; lane 2: negative control; lane M: Marker II (Tiangen). (B) Number of eggs laid by each female placed in a clip-cage attached to a leaf of a healthy or a TYLCV-infected tomato plant. Values are means ± SE of 10 replicate plants (three adults per plant). (C) The OD value of TSWV analysis obtained by DAS-ELISA indicated the difference between F. occidentalis that were exposed and not exposed to TYLCV. The number in blue indicates the mean OD value of healthy tomato plants. Values are means ± SE. For (B,C) , different letters indicate significant differences between treatments ( P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Acquisition of TYLCV by Frankliniella occidentalis (A) , fecundity of F. occidentalis as affected by TYLCV (B) , and transmission of TSWV by F. occidentalis as affected by previous exposure of the thrips to TYLCV (C) . (A) PCR amplification of TYLCV was performed with the DNA extracted from F. occidentalis second-instar nymphs (lanes 3–7; one nymph per lane) and newly emerged adults (lanes 8–12; one adult per lane) obtained from a TYLCV-infected tomato plant. Amplification products were visualized by agarose gel electrophoresis with Gelview staining. Lane 1: positive control; lane 2: negative control; lane M: Marker II (Tiangen). (B) Number of eggs laid by each female placed in a clip-cage attached to a leaf of a healthy or a TYLCV-infected tomato plant. Values are means ± SE of 10 replicate plants (three adults per plant). (C) The OD value of TSWV analysis obtained by DAS-ELISA indicated the difference between F. occidentalis that were exposed and not exposed to TYLCV. The number in blue indicates the mean OD value of healthy tomato plants. Values are means ± SE. For (B,C) , different letters indicate significant differences between treatments ( P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques: Transmission Assay, Polymerase Chain Reaction, Amplification, Infection, Agarose Gel Electrophoresis, Staining, Positive Control, Negative Control, Marker, Cross-linking Immunoprecipitation, Enzyme-linked Immunosorbent Assay

    Acquisition of TYLCV DNA by Bemisia tabaci Q females as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h acquisition access period (AAP) on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on TYLCV-infected tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗ P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Acquisition of TYLCV DNA by Bemisia tabaci Q females as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h acquisition access period (AAP) on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on TYLCV-infected tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗ P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques: Infection

    PCR detection of 22 putative transgenic plants. 1~ 22 are putative transgenic plants, positive control (plasmid DNA as template, CK+) and negative control (non-transgenic plants, CK-)

    Journal: Hereditas

    Article Title: Enhancement of grain number per spike by RNA interference of cytokinin oxidase 2 gene in bread wheat

    doi: 10.1186/s41065-018-0071-7

    Figure Lengend Snippet: PCR detection of 22 putative transgenic plants. 1~ 22 are putative transgenic plants, positive control (plasmid DNA as template, CK+) and negative control (non-transgenic plants, CK-)

    Article Snippet: Genomic DNA was extracted from young leaf tissues of receptor NB1 and transgenic plants using plant genomic DNA Extraction Kit (Tiangen Biotech, Beijing, China).

    Techniques: Polymerase Chain Reaction, Transgenic Assay, Positive Control, Plasmid Preparation, Negative Control

    Southern blotting analysis in selected T 0 primary wheat transformants. Genomic DNA digested with Eco R V and hybridized with FAD2 probe. The number of reactive bands in each lane represents the transgene copies in each transgenic line. Lane 1 is WT (NB1), lanes 2~ 6 are JW1-1A, JW5-1A, JW41-1B, JW39-3A and JW1-2B, respectively, M is the marker: λ-EcoT14 I digest (TaKaRa, Dalian, China)

    Journal: Hereditas

    Article Title: Enhancement of grain number per spike by RNA interference of cytokinin oxidase 2 gene in bread wheat

    doi: 10.1186/s41065-018-0071-7

    Figure Lengend Snippet: Southern blotting analysis in selected T 0 primary wheat transformants. Genomic DNA digested with Eco R V and hybridized with FAD2 probe. The number of reactive bands in each lane represents the transgene copies in each transgenic line. Lane 1 is WT (NB1), lanes 2~ 6 are JW1-1A, JW5-1A, JW41-1B, JW39-3A and JW1-2B, respectively, M is the marker: λ-EcoT14 I digest (TaKaRa, Dalian, China)

    Article Snippet: Genomic DNA was extracted from young leaf tissues of receptor NB1 and transgenic plants using plant genomic DNA Extraction Kit (Tiangen Biotech, Beijing, China).

    Techniques: Southern Blot, Transgenic Assay, Marker

    The ChIP assay of S. cellulosum So ce M4 containing recombinant TALE-VP64 vector: (A) the SDS-PAGE analysis of ChIP eluate; (B) the Western blot analysis of ChIP eluate; (C) the verification of the presence of P3 promoter in ChIP eluate: M. DNA marker, lanes 1–3 were positive control, blank control, and PCR product using ChIP eluate as a template.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: An Easy and Efficient Strategy for the Enhancement of Epothilone Production Mediated by TALE-TF and CRISPR/dcas9 Systems in Sorangium cellulosum

    doi: 10.3389/fbioe.2019.00334

    Figure Lengend Snippet: The ChIP assay of S. cellulosum So ce M4 containing recombinant TALE-VP64 vector: (A) the SDS-PAGE analysis of ChIP eluate; (B) the Western blot analysis of ChIP eluate; (C) the verification of the presence of P3 promoter in ChIP eluate: M. DNA marker, lanes 1–3 were positive control, blank control, and PCR product using ChIP eluate as a template.

    Article Snippet: The genomic DNAs of recombinant So ce M4 clones were extracted using the Genomic DNA extraction kit (Tiangen, Beijing, China) and then used as templates; the primers colE F and colE R were used to amplify colE replicon in Ptalen R36 vector to ensure the introduction of recombinant TALE-TF vector.

    Techniques: Chromatin Immunoprecipitation, Recombinant, Plasmid Preparation, SDS Page, Western Blot, Marker, Positive Control, Polymerase Chain Reaction

    The construction and introduction of recombinant TALE-VP64 and dCas9-VP64 elements: (A) the insertion of VP64 element: M. DNA Marker; lanes 1–11 were colony PCR products, lane 9 was the positive clone; (B) the insertion of P43 promoter into TALE-VP64 vector and pLX-sgRNA, lanes 1–4 were colony PCR using different colonies as template, lane 5 was positive control; (C) the confirmation of the introduction of TALE-VP64-P3 into So ce M4, lane 1 was the native So ce M4, lanes 2–6 were colony PCR products; (D) the confirmation of the introduction of dCas9-VP64-P3 into So ce M4: lane 1 was the native So ce M4, lanes 2 and 3 were colony PCR products; (E) the confirmation of the introduction of TALE-VP64-P3 vector by Western blot, anti-FLAG antibody was used as a primary antibody.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: An Easy and Efficient Strategy for the Enhancement of Epothilone Production Mediated by TALE-TF and CRISPR/dcas9 Systems in Sorangium cellulosum

    doi: 10.3389/fbioe.2019.00334

    Figure Lengend Snippet: The construction and introduction of recombinant TALE-VP64 and dCas9-VP64 elements: (A) the insertion of VP64 element: M. DNA Marker; lanes 1–11 were colony PCR products, lane 9 was the positive clone; (B) the insertion of P43 promoter into TALE-VP64 vector and pLX-sgRNA, lanes 1–4 were colony PCR using different colonies as template, lane 5 was positive control; (C) the confirmation of the introduction of TALE-VP64-P3 into So ce M4, lane 1 was the native So ce M4, lanes 2–6 were colony PCR products; (D) the confirmation of the introduction of dCas9-VP64-P3 into So ce M4: lane 1 was the native So ce M4, lanes 2 and 3 were colony PCR products; (E) the confirmation of the introduction of TALE-VP64-P3 vector by Western blot, anti-FLAG antibody was used as a primary antibody.

    Article Snippet: The genomic DNAs of recombinant So ce M4 clones were extracted using the Genomic DNA extraction kit (Tiangen, Beijing, China) and then used as templates; the primers colE F and colE R were used to amplify colE replicon in Ptalen R36 vector to ensure the introduction of recombinant TALE-TF vector.

    Techniques: Recombinant, Marker, Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Western Blot

    Vaccination with CpG adjuvant reduces HBV DNA in the liver. ( a ) AAV/HBV (2×10 10 vg)-infected mice were treated with the indicated vaccine combinations. HBV DNA levels in the liver were determined by real-time PCR at 11 weeks post-vaccination. ( b ) HBsAg levels in the liver were measured by ELISA at 11 weeks post-vaccination with homogenized liver tissues. ( c ) Immunohistochemical staining of HBcAg in the liver sections of different groups ( n =3). AAV, adeno-associated virus; HBcAg, HBV core antigen; HBsAg, HBV surface antigen; HBV, hepatitis B virus.

    Journal: Cellular and Molecular Immunology

    Article Title: A mouse model for HBV immunotolerance and immunotherapy

    doi: 10.1038/cmi.2013.43

    Figure Lengend Snippet: Vaccination with CpG adjuvant reduces HBV DNA in the liver. ( a ) AAV/HBV (2×10 10 vg)-infected mice were treated with the indicated vaccine combinations. HBV DNA levels in the liver were determined by real-time PCR at 11 weeks post-vaccination. ( b ) HBsAg levels in the liver were measured by ELISA at 11 weeks post-vaccination with homogenized liver tissues. ( c ) Immunohistochemical staining of HBcAg in the liver sections of different groups ( n =3). AAV, adeno-associated virus; HBcAg, HBV core antigen; HBsAg, HBV surface antigen; HBV, hepatitis B virus.

    Article Snippet: Liver HBV DNA was extracted from 50 mg of liver tissues using a genomic DNA kit (TIANGEN Biotech, Beijing, China).

    Techniques: Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

    AAV/HBV inoculation resulted in persistent HBV viremia in immunocompetent mice. ( a ) Female or male C57BL/6 mice ( n =6, 6–8 weeks old) were infected with AAV/HBV at 5×10 10 viral genome equivalents (vg) through tail vein injection. At 14 days post-infection, blood samples were collected and serum HBsAg was measured by ELISA. ( b ) Intraliver injection of 3-day-old neonatal C57BL/6 mice ( n =6–7) was performed with 1×10 10 or 2×10 10 vg AAV/HBV, and serum HBsAg titers were examined at 48 days post-infection. ( c ) C57BL/6 mice ( n =6, 6–8 weeks old) were infected with AAV/HBV at 2×10 10 , 5×10 10 or 1×10 11 vg. The mice were bled biweekly after infection. Serum HBsAg was measured by ELISA. The lower limit of detection was 0.5 ng/ml. ( d ) Adult C57BL/6 mice were intravenously injected with 2×10 10 , 5×10 10 or 1×10 11 vg virus and were bled biweekly to monitor serum HBeAg titers. ( e ) On day 40 post-AAV/HBV infection, HBV genomic DNA in the serum was determined by real-time PCR. ( f ) Immunohistochemical staining of HBcAg in the liver of AAV/HBV infected mice (12 weeks post-infection). AAV, adeno-associated virus; HBcAg, HBV core antigen; HBsAg, HBV surface antigen; HBV, hepatitis B virus.

    Journal: Cellular and Molecular Immunology

    Article Title: A mouse model for HBV immunotolerance and immunotherapy

    doi: 10.1038/cmi.2013.43

    Figure Lengend Snippet: AAV/HBV inoculation resulted in persistent HBV viremia in immunocompetent mice. ( a ) Female or male C57BL/6 mice ( n =6, 6–8 weeks old) were infected with AAV/HBV at 5×10 10 viral genome equivalents (vg) through tail vein injection. At 14 days post-infection, blood samples were collected and serum HBsAg was measured by ELISA. ( b ) Intraliver injection of 3-day-old neonatal C57BL/6 mice ( n =6–7) was performed with 1×10 10 or 2×10 10 vg AAV/HBV, and serum HBsAg titers were examined at 48 days post-infection. ( c ) C57BL/6 mice ( n =6, 6–8 weeks old) were infected with AAV/HBV at 2×10 10 , 5×10 10 or 1×10 11 vg. The mice were bled biweekly after infection. Serum HBsAg was measured by ELISA. The lower limit of detection was 0.5 ng/ml. ( d ) Adult C57BL/6 mice were intravenously injected with 2×10 10 , 5×10 10 or 1×10 11 vg virus and were bled biweekly to monitor serum HBeAg titers. ( e ) On day 40 post-AAV/HBV infection, HBV genomic DNA in the serum was determined by real-time PCR. ( f ) Immunohistochemical staining of HBcAg in the liver of AAV/HBV infected mice (12 weeks post-infection). AAV, adeno-associated virus; HBcAg, HBV core antigen; HBsAg, HBV surface antigen; HBV, hepatitis B virus.

    Article Snippet: Liver HBV DNA was extracted from 50 mg of liver tissues using a genomic DNA kit (TIANGEN Biotech, Beijing, China).

    Techniques: Mouse Assay, Infection, Injection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining

    The immune response was undetectable in AAV/HBV-infected mice. ( a ) Serum HBsAb levels in C57BL/6 mice receiving 15 µg of pBlue-HBV1.3 DNA via hydrodynamic injection or infected with 1×10 11 vg of AAV/HBV ( n =3). ( b ) ALT levels of HBV carrier mice were measured over time ( n =3). ( c ) H E staining of liver sections from mice infected with different doses of AAV/HBV. One of two independent experiments is presented. AAV, adeno-associated virus; ALT, alanine aminotransferase; HBsAg, HBV surface antigen; HBV, hepatitis B virus; H E, hematoxylin and eosin.

    Journal: Cellular and Molecular Immunology

    Article Title: A mouse model for HBV immunotolerance and immunotherapy

    doi: 10.1038/cmi.2013.43

    Figure Lengend Snippet: The immune response was undetectable in AAV/HBV-infected mice. ( a ) Serum HBsAb levels in C57BL/6 mice receiving 15 µg of pBlue-HBV1.3 DNA via hydrodynamic injection or infected with 1×10 11 vg of AAV/HBV ( n =3). ( b ) ALT levels of HBV carrier mice were measured over time ( n =3). ( c ) H E staining of liver sections from mice infected with different doses of AAV/HBV. One of two independent experiments is presented. AAV, adeno-associated virus; ALT, alanine aminotransferase; HBsAg, HBV surface antigen; HBV, hepatitis B virus; H E, hematoxylin and eosin.

    Article Snippet: Liver HBV DNA was extracted from 50 mg of liver tissues using a genomic DNA kit (TIANGEN Biotech, Beijing, China).

    Techniques: Infection, Mouse Assay, Injection, Staining

    DNA sequencing analysis of the large DNA fragment (mutant hlyC , 3072-bp) amplified with hlyC flanking primers ( P11 , Table 1 ) from DNA of M . hyorhinis cells transformed with Mini-oriC-HT1 ( A ) and Mini-oriC-HT2 ( B ) targeting plasmids. The sequencing was performed in both forward and reverse directions using hlyC flanking primers, P11 (Table 1 )”

    Journal: Scientific Reports

    Article Title: Development of oriC-plasmids for use in Mycoplasma hyorhinis

    doi: 10.1038/s41598-017-10519-3

    Figure Lengend Snippet: DNA sequencing analysis of the large DNA fragment (mutant hlyC , 3072-bp) amplified with hlyC flanking primers ( P11 , Table 1 ) from DNA of M . hyorhinis cells transformed with Mini-oriC-HT1 ( A ) and Mini-oriC-HT2 ( B ) targeting plasmids. The sequencing was performed in both forward and reverse directions using hlyC flanking primers, P11 (Table 1 )”

    Article Snippet: To detect the presence of the plasmids in the transformants, total DNA was extracted from 2 ml M . hyorhinis cultures using a genomic DNA purification kit (TIANGEN, Beijing, China).

    Techniques: DNA Sequencing, Mutagenesis, Amplification, Transformation Assay, Sequencing

    Analysis of hlyC disruption: DNA was extracted from the grown culture of M . hyorhinis transformed with Mini-oriC-HT1 ( A ) and Mini-oriC-HT2 ( B ) targeting plasmids along with control untransformed cultures, was subjected to PCR analysis using hlyC flanking primers ( P11 , Table 1 ) to investigate the integration of the tetM into M . hyorhinis genome at hlyC site. Wild-type hemolysin exhibits 1566-bp while mutant hemolysin that encodes tetM along with spiralin gene promoter has about 3072-bp. ( C ) the phenotype of the wild-type and mutant colonies of M . hyorhinis . The gel image was cropped and full-length gel is included in the Supplementary Fig. S10 .

    Journal: Scientific Reports

    Article Title: Development of oriC-plasmids for use in Mycoplasma hyorhinis

    doi: 10.1038/s41598-017-10519-3

    Figure Lengend Snippet: Analysis of hlyC disruption: DNA was extracted from the grown culture of M . hyorhinis transformed with Mini-oriC-HT1 ( A ) and Mini-oriC-HT2 ( B ) targeting plasmids along with control untransformed cultures, was subjected to PCR analysis using hlyC flanking primers ( P11 , Table 1 ) to investigate the integration of the tetM into M . hyorhinis genome at hlyC site. Wild-type hemolysin exhibits 1566-bp while mutant hemolysin that encodes tetM along with spiralin gene promoter has about 3072-bp. ( C ) the phenotype of the wild-type and mutant colonies of M . hyorhinis . The gel image was cropped and full-length gel is included in the Supplementary Fig. S10 .

    Article Snippet: To detect the presence of the plasmids in the transformants, total DNA was extracted from 2 ml M . hyorhinis cultures using a genomic DNA purification kit (TIANGEN, Beijing, China).

    Techniques: Transformation Assay, Polymerase Chain Reaction, Mutagenesis

    Growth of Hsp90 knockout parasite ( ΔHsp90 ) in vitro. The parasites were cultured in African green monkey kidney (Vero) cells, 10 5 T. gondii were added to the 6-well plates, and the infection ratio was 1:1. Observation of RH Δku80 (A), ΔHsp90 (B), and complemented (C) parasites by inverted microscope. The plaque produced by ΔPKAR strains (Fig. 6B) was significantly smaller than that of RH Δku80 and complemented parasites (Figs. 6A, 6C), scale bar = 20 μm. T. gondii tachyzoites and Vero cells were indicated by arrows, T. gondii (black arrow), Vero cells (white arrow). (D) The parasites were collected at the same time, and genomic DNA was extracted by TIANGEN kit. T. gondii DNA was detected by SYBR-green real-time PCR using B1 primer pairs, the standard curve was obtained by the known concentration of the RH Δku80 parasites with the primers (B1), and the parasite number was calculated by interpolation from this standard curve. ** p

    Journal: Parasite

    Article Title: The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth

    doi: 10.1051/parasite/2017023

    Figure Lengend Snippet: Growth of Hsp90 knockout parasite ( ΔHsp90 ) in vitro. The parasites were cultured in African green monkey kidney (Vero) cells, 10 5 T. gondii were added to the 6-well plates, and the infection ratio was 1:1. Observation of RH Δku80 (A), ΔHsp90 (B), and complemented (C) parasites by inverted microscope. The plaque produced by ΔPKAR strains (Fig. 6B) was significantly smaller than that of RH Δku80 and complemented parasites (Figs. 6A, 6C), scale bar = 20 μm. T. gondii tachyzoites and Vero cells were indicated by arrows, T. gondii (black arrow), Vero cells (white arrow). (D) The parasites were collected at the same time, and genomic DNA was extracted by TIANGEN kit. T. gondii DNA was detected by SYBR-green real-time PCR using B1 primer pairs, the standard curve was obtained by the known concentration of the RH Δku80 parasites with the primers (B1), and the parasite number was calculated by interpolation from this standard curve. ** p

    Article Snippet: Next, 24, 48, 72, and 96 h post-infection (PI), the parasites were collected and genomic DNA was extracted using the TIANGEN genomic DNA isolation kit by following the manufacturer’s protocol (TIANGEN, Beijing).

    Techniques: Knock-Out, In Vitro, Cell Culture, Infection, Inverted Microscopy, Produced, SYBR Green Assay, Real-time Polymerase Chain Reaction, Concentration Assay

    Confirmation of ccpN point mutation and ccpN in-frame deletion by PCR. The PCR product was analyzed by agarose gel electrophoresis. A 1(Fermentas) was used as a molecular weight marker (lane M). A. Confirmation of the ccpN point mutation. Fragments were amplified using ccpN-Mut-P7/ccpN-Mut-P8 as primers. Each lane showed amplified DNA generated from a DNA template: lane 1, BUK-1C (Δ ccpN :: upp -cassette); lane 2, BUK-1 ( ccpN -mut-Ala 130 to Ser); lane 3, dsDNA PCR fragment (positive control); lane 4, BUK ( ccpN -wild type) (negative control). B. Confirmation of the ccpN in-frame deletion. Fragments were amplified using ccpN-Del-P1/ccpN-Del-P6 as primers. Each lane showed amplified DNA generated from a DNA template: lane 5, BUK-2C (Δ ccpN :: upp -cassette); lane 6, BUK-2 (Δ ccpN ); lane 7, dsDNA PCR fragment (positive control); lane 8, BUK ( ccpN -wild type) (negative control).

    Journal: PLoS ONE

    Article Title: Establishment of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a Double-Strand Break in the Chromosome

    doi: 10.1371/journal.pone.0081370

    Figure Lengend Snippet: Confirmation of ccpN point mutation and ccpN in-frame deletion by PCR. The PCR product was analyzed by agarose gel electrophoresis. A 1(Fermentas) was used as a molecular weight marker (lane M). A. Confirmation of the ccpN point mutation. Fragments were amplified using ccpN-Mut-P7/ccpN-Mut-P8 as primers. Each lane showed amplified DNA generated from a DNA template: lane 1, BUK-1C (Δ ccpN :: upp -cassette); lane 2, BUK-1 ( ccpN -mut-Ala 130 to Ser); lane 3, dsDNA PCR fragment (positive control); lane 4, BUK ( ccpN -wild type) (negative control). B. Confirmation of the ccpN in-frame deletion. Fragments were amplified using ccpN-Del-P1/ccpN-Del-P6 as primers. Each lane showed amplified DNA generated from a DNA template: lane 5, BUK-2C (Δ ccpN :: upp -cassette); lane 6, BUK-2 (Δ ccpN ); lane 7, dsDNA PCR fragment (positive control); lane 8, BUK ( ccpN -wild type) (negative control).

    Article Snippet: Confirmation of Mutants by PCR and Sequencing To confirm the desired mutations, genome DNA was isolated from cells using the TIANamp Bacteria DNA Kit (Tiangen, Beijing, China) following the manufacturer’s instructions.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Marker, Amplification, Generated, Positive Control, Negative Control

    Markerless deletion of large chromosomal regions stimulated by DSB. A. (a) Two dsDNA fragments were generated by fusion PCR and integrated into genome by double-crossover events. (b) The endonuclease I-S ce I was expressed from pEBS- cop 1 under the induction of xylose, and the genome of B. subtlis BUK-II was subjected to I-S ce I cleavage. (c) Deletion mutants were isolated on MM plate containing 10 µM 5FU and confirmed by PCR. A, B, C, D represent DNA segments selected for homologous recombination. B. Verification of the 20.5 kb and 75.9 kb deletions in BUK by PCR. Fragments were amplified using the primer pairs 20.5 kb-DEL-P15/20.5 kb-DEL-P16 (a), 20.5 kb-DEL-P17/20.5 kb-DEL-P18 (b), 75.9 kb-DEL-P15/75.9 kb-DEL-P16 (c), 75.9 kb-DEL-P17/75.9 kb-DEL-P18 (d). 1 kb DNA Ladder (Fermentas) was used as a molecular weight marker (lane M). (a) Amplification of the ydcR gene (534334–535389 SubtiList coordinates) in the 20.5 kb DNA region of B . subtilis ; (b) Amplification of the 20.5 kb DNA fragment of B. subtilis ; (c–d) Confirmation of the 75.9 kb deletion of BUK was the same as that of 20.5 kb deletion. Amplification of the pksG gene (1789763–1791405 SubtiList coordinates) in the 75.9 kb DNA region of B . subtilis (c) and amplification of the 75.9 kb DNA fragment of B. subtilis (d). Each lane showed amplified DNA generated from a DNA template: lane 1, BUKΔ20.5 kb; lane 2, BUK-II (20.5); lane 3, BUK; lane 4, BUKΔ75.9 kb; lane 5, BUK-II (76.9); lane 6, BUK.

    Journal: PLoS ONE

    Article Title: Establishment of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a Double-Strand Break in the Chromosome

    doi: 10.1371/journal.pone.0081370

    Figure Lengend Snippet: Markerless deletion of large chromosomal regions stimulated by DSB. A. (a) Two dsDNA fragments were generated by fusion PCR and integrated into genome by double-crossover events. (b) The endonuclease I-S ce I was expressed from pEBS- cop 1 under the induction of xylose, and the genome of B. subtlis BUK-II was subjected to I-S ce I cleavage. (c) Deletion mutants were isolated on MM plate containing 10 µM 5FU and confirmed by PCR. A, B, C, D represent DNA segments selected for homologous recombination. B. Verification of the 20.5 kb and 75.9 kb deletions in BUK by PCR. Fragments were amplified using the primer pairs 20.5 kb-DEL-P15/20.5 kb-DEL-P16 (a), 20.5 kb-DEL-P17/20.5 kb-DEL-P18 (b), 75.9 kb-DEL-P15/75.9 kb-DEL-P16 (c), 75.9 kb-DEL-P17/75.9 kb-DEL-P18 (d). 1 kb DNA Ladder (Fermentas) was used as a molecular weight marker (lane M). (a) Amplification of the ydcR gene (534334–535389 SubtiList coordinates) in the 20.5 kb DNA region of B . subtilis ; (b) Amplification of the 20.5 kb DNA fragment of B. subtilis ; (c–d) Confirmation of the 75.9 kb deletion of BUK was the same as that of 20.5 kb deletion. Amplification of the pksG gene (1789763–1791405 SubtiList coordinates) in the 75.9 kb DNA region of B . subtilis (c) and amplification of the 75.9 kb DNA fragment of B. subtilis (d). Each lane showed amplified DNA generated from a DNA template: lane 1, BUKΔ20.5 kb; lane 2, BUK-II (20.5); lane 3, BUK; lane 4, BUKΔ75.9 kb; lane 5, BUK-II (76.9); lane 6, BUK.

    Article Snippet: Confirmation of Mutants by PCR and Sequencing To confirm the desired mutations, genome DNA was isolated from cells using the TIANamp Bacteria DNA Kit (Tiangen, Beijing, China) following the manufacturer’s instructions.

    Techniques: Generated, Polymerase Chain Reaction, Isolation, Homologous Recombination, Amplification, Molecular Weight, Marker

    Characterization of B. amyloliquefaciens strain HAB-2 and its mutant. a Gel electrophoresis of PCR products of wild-type strain HAB-2 (lane HAB-2) and its mutant (lane Mutant); b MALDI-TOF MS analysis on the extract of HAB△ lpa mutant, 3000 bp DNA marker

    Journal: BMC Microbiology

    Article Title: Characterization of lpaH2 gene corresponding to lipopeptide synthesis in Bacillus amyloliquefaciens HAB-2

    doi: 10.1186/s12866-017-1134-z

    Figure Lengend Snippet: Characterization of B. amyloliquefaciens strain HAB-2 and its mutant. a Gel electrophoresis of PCR products of wild-type strain HAB-2 (lane HAB-2) and its mutant (lane Mutant); b MALDI-TOF MS analysis on the extract of HAB△ lpa mutant, 3000 bp DNA marker

    Article Snippet: The chromosomal DNA of strain HAB-2 was extracted using Bacterial Genomic DNA Extraction Kit (Tiangen Biochemical Science and Technology Co., LTD) according to the manufacturer’s protocol.

    Techniques: Mutagenesis, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Mass Spectrometry, Marker

    Lipopeptide analysis from B. amyloliquefaciens HAB-2 and B. subtilis 168. a Amplification of lpa and sfp gene fragments from B. amyloliquefaciens HAB-2 and B. subtilis 168. From lane 1 to 4: HAB-2- sfp ; B. subtilis 168- sfp ; HAB-2- lpa ; and B. subtilis 168- lpa . Lane M: 2000 bp DNA marker. b Southern-blot hybridization for lpaH2 and sfp genes, a: lpa from the strain HAB-2, b: chromosomal DNA of the strain HAB-2, c: chromosomal DNA of B. subtilis 168, d: sfp from B. subtilis 168, e: chromosomal DNA of B. subtilis 168, f: chromosomal DNA of the strain HAB-2. c Antifungal activity of B. amyloliquefaciens HAB-2 and B. subtilis 168 against C. gloeosporioides on agar plate. d Inhibitory effect of lipopetides produced from B. amyloliquefaciens HAB-2 against C. gloeosporioides

    Journal: BMC Microbiology

    Article Title: Characterization of lpaH2 gene corresponding to lipopeptide synthesis in Bacillus amyloliquefaciens HAB-2

    doi: 10.1186/s12866-017-1134-z

    Figure Lengend Snippet: Lipopeptide analysis from B. amyloliquefaciens HAB-2 and B. subtilis 168. a Amplification of lpa and sfp gene fragments from B. amyloliquefaciens HAB-2 and B. subtilis 168. From lane 1 to 4: HAB-2- sfp ; B. subtilis 168- sfp ; HAB-2- lpa ; and B. subtilis 168- lpa . Lane M: 2000 bp DNA marker. b Southern-blot hybridization for lpaH2 and sfp genes, a: lpa from the strain HAB-2, b: chromosomal DNA of the strain HAB-2, c: chromosomal DNA of B. subtilis 168, d: sfp from B. subtilis 168, e: chromosomal DNA of B. subtilis 168, f: chromosomal DNA of the strain HAB-2. c Antifungal activity of B. amyloliquefaciens HAB-2 and B. subtilis 168 against C. gloeosporioides on agar plate. d Inhibitory effect of lipopetides produced from B. amyloliquefaciens HAB-2 against C. gloeosporioides

    Article Snippet: The chromosomal DNA of strain HAB-2 was extracted using Bacterial Genomic DNA Extraction Kit (Tiangen Biochemical Science and Technology Co., LTD) according to the manufacturer’s protocol.

    Techniques: Amplification, Marker, Southern Blot, Hybridization, Activity Assay, Produced