t7 endonuclease Search Results


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    New England Biolabs t7 endonuclease
    The anti-HCMV CRISPR/Cas9 system induces mutations resulting in a decrease in IE protein expression in primary fibroblasts. MRC5 cells were transduced with one of the three type 1 LVs and selected by puromycin treatment (2 μg/mL) for 2 days. Control (untransduced) and puromycin-resistant MRC5 cells were subcultured prior to infection with Toledo (MOI of 0.1). Two days pi, proteins and DNA were extracted from the infected cells via TriPrep Kit. a) Viral DNA extracts were PCR-amplified at the target region. Amplicons were subsequently subjected to <t>T7</t> endonuclease to detect the indels induced by the singleplex strategy. b) PCR amplicons of the whole IE gene were analyzed to detect larger deletions induced by the multiplex strategy. The arrows highlight the indels (singleplex) and larger deletions (multiplex) induced by the anti-HCMV CRISPR/Cas9 strategies (one out of three independent experiments is shown). c) Western blot analysis of IE and Cas9 expression 2 days pi (one representative western blot out of 3 independent experiments is shown). d) At each passage, the proteins were extracted with the TriPrep Kit and the Cas9 expression was assessed by Western blot. e) Relative quantification of Cas9 expression based on the Western blot (d) normalized to the housekeeping protein actin. Pt, post-transduction.
    T7 Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 904 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs dnase i
    Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of <t>DNase</t> I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p
    Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8698 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The anti-HCMV CRISPR/Cas9 system induces mutations resulting in a decrease in IE protein expression in primary fibroblasts. MRC5 cells were transduced with one of the three type 1 LVs and selected by puromycin treatment (2 μg/mL) for 2 days. Control (untransduced) and puromycin-resistant MRC5 cells were subcultured prior to infection with Toledo (MOI of 0.1). Two days pi, proteins and DNA were extracted from the infected cells via TriPrep Kit. a) Viral DNA extracts were PCR-amplified at the target region. Amplicons were subsequently subjected to T7 endonuclease to detect the indels induced by the singleplex strategy. b) PCR amplicons of the whole IE gene were analyzed to detect larger deletions induced by the multiplex strategy. The arrows highlight the indels (singleplex) and larger deletions (multiplex) induced by the anti-HCMV CRISPR/Cas9 strategies (one out of three independent experiments is shown). c) Western blot analysis of IE and Cas9 expression 2 days pi (one representative western blot out of 3 independent experiments is shown). d) At each passage, the proteins were extracted with the TriPrep Kit and the Cas9 expression was assessed by Western blot. e) Relative quantification of Cas9 expression based on the Western blot (d) normalized to the housekeeping protein actin. Pt, post-transduction.

    Journal: PLoS ONE

    Article Title: Multiplex CRISPR/Cas9 system impairs HCMV replication by excising an essential viral gene

    doi: 10.1371/journal.pone.0192602

    Figure Lengend Snippet: The anti-HCMV CRISPR/Cas9 system induces mutations resulting in a decrease in IE protein expression in primary fibroblasts. MRC5 cells were transduced with one of the three type 1 LVs and selected by puromycin treatment (2 μg/mL) for 2 days. Control (untransduced) and puromycin-resistant MRC5 cells were subcultured prior to infection with Toledo (MOI of 0.1). Two days pi, proteins and DNA were extracted from the infected cells via TriPrep Kit. a) Viral DNA extracts were PCR-amplified at the target region. Amplicons were subsequently subjected to T7 endonuclease to detect the indels induced by the singleplex strategy. b) PCR amplicons of the whole IE gene were analyzed to detect larger deletions induced by the multiplex strategy. The arrows highlight the indels (singleplex) and larger deletions (multiplex) induced by the anti-HCMV CRISPR/Cas9 strategies (one out of three independent experiments is shown). c) Western blot analysis of IE and Cas9 expression 2 days pi (one representative western blot out of 3 independent experiments is shown). d) At each passage, the proteins were extracted with the TriPrep Kit and the Cas9 expression was assessed by Western blot. e) Relative quantification of Cas9 expression based on the Western blot (d) normalized to the housekeeping protein actin. Pt, post-transduction.

    Article Snippet: Two hundred nanograms of purified PCR products was denatured for five minutes at 95 °C and slowly re-annealed using three steps consisting of 15 s at 95 °C, 15 s at 85 °C and 30 s at 25 °C, followed by a 30 min digestion at 37 °C with T7 endonuclease (New England Biolab Inc., UK).

    Techniques: CRISPR, Expressing, Transduction, Infection, Polymerase Chain Reaction, Amplification, Multiplex Assay, Western Blot

    Surrogate reporter-mediated magnetic separations enrich ZFN- and TALEN-driven mutant cells. Nuclease-driven mutations were detected by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gels indicate mutation percentages calculated by band intensities.

    Journal: PLoS ONE

    Article Title: Magnetic Separation and Antibiotics Selection Enable Enrichment of Cells with ZFN/TALEN-Induced Mutations

    doi: 10.1371/journal.pone.0056476

    Figure Lengend Snippet: Surrogate reporter-mediated magnetic separations enrich ZFN- and TALEN-driven mutant cells. Nuclease-driven mutations were detected by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gels indicate mutation percentages calculated by band intensities.

    Article Snippet: The amplicons were denatured by heating and annealed to form heteroduplex DNA, which was treated with 5 units of T7 endonuclease 1 (New England Biolabs) for 15 to 20 min at 37°C and then analyzed by 2% agarose gel electrophoresis.

    Techniques: Mutagenesis

    Hygromycin selection through use of a surrogate reporter system enriches nuclease-induced mutant cells. (A) Enrichment of GFP + cells after hygromycin selection. Scale bar = 50 µm. (B) ZFN-driven mutations detected by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. (C) DNA sequences of the wild-type (WT) and mutant clones, with ZFN recognition sites underlined. Dashes indicate deleted bases, and small bold letters indicate inserted bases. The number of occurrences is shown in parentheses; X1, X2, and X3 indicate the number of times that each clone was detected. Mutation frequencies were calculated by dividing the number of mutant clones by the number of total clones.

    Journal: PLoS ONE

    Article Title: Magnetic Separation and Antibiotics Selection Enable Enrichment of Cells with ZFN/TALEN-Induced Mutations

    doi: 10.1371/journal.pone.0056476

    Figure Lengend Snippet: Hygromycin selection through use of a surrogate reporter system enriches nuclease-induced mutant cells. (A) Enrichment of GFP + cells after hygromycin selection. Scale bar = 50 µm. (B) ZFN-driven mutations detected by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. (C) DNA sequences of the wild-type (WT) and mutant clones, with ZFN recognition sites underlined. Dashes indicate deleted bases, and small bold letters indicate inserted bases. The number of occurrences is shown in parentheses; X1, X2, and X3 indicate the number of times that each clone was detected. Mutation frequencies were calculated by dividing the number of mutant clones by the number of total clones.

    Article Snippet: The amplicons were denatured by heating and annealed to form heteroduplex DNA, which was treated with 5 units of T7 endonuclease 1 (New England Biolabs) for 15 to 20 min at 37°C and then analyzed by 2% agarose gel electrophoresis.

    Techniques: Selection, Mutagenesis, Clone Assay

    Magnetic separation-mediated enrichment of CCR5 -disrupted cells using an episomal reporter. (A) Enrichment of GFP + cells after magnetic separation. Scale bar = 50 µm. (B) ZFN-driven mutations detected by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. (C) DNA sequences of the wild-type (WT) and mutant clones, with ZFN recognition sites underlined. Dashes indicate deleted bases, and small bold letters indicate inserted bases. The number of occurrences is shown in parentheses; X1 indicates that each clone was detected once. Mutation frequencies were calculated by dividing the number of mutant clones by the number of total clones.

    Journal: PLoS ONE

    Article Title: Magnetic Separation and Antibiotics Selection Enable Enrichment of Cells with ZFN/TALEN-Induced Mutations

    doi: 10.1371/journal.pone.0056476

    Figure Lengend Snippet: Magnetic separation-mediated enrichment of CCR5 -disrupted cells using an episomal reporter. (A) Enrichment of GFP + cells after magnetic separation. Scale bar = 50 µm. (B) ZFN-driven mutations detected by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. (C) DNA sequences of the wild-type (WT) and mutant clones, with ZFN recognition sites underlined. Dashes indicate deleted bases, and small bold letters indicate inserted bases. The number of occurrences is shown in parentheses; X1 indicates that each clone was detected once. Mutation frequencies were calculated by dividing the number of mutant clones by the number of total clones.

    Article Snippet: The amplicons were denatured by heating and annealed to form heteroduplex DNA, which was treated with 5 units of T7 endonuclease 1 (New England Biolabs) for 15 to 20 min at 37°C and then analyzed by 2% agarose gel electrophoresis.

    Techniques: Mutagenesis, Clone Assay

    Proof of Concept of Anti-HBV Efficacy of TALENs or CRISPR/Cas9 (A) HEK293 cells were co-transfected with an HBV expression plasmid pTHBV2 and either of the complete nuclease systems. DNA was isolated after 4 days and examined for mutagenesis at target sites by T7E1 assay. Undigested and digested products were separated on an agarose gel side by side for comparison. Expected cleavage product sizes are indicated by arrowheads. “M” indicates the molecular weight marker lane. (B) Huh7 cells were co-transfected with an HBV expression plasmid pTHBV2 and TALEN or CRISPR/Cas9 nuclease systems. HBsAg concentrations in the supernatant were measured after 4 days by ELISA. Data are represented as means of S/CO (sample to control ratio) values and error bars indicate SD of three replicates. Statistically significant differences to HBV only are indicated by an asterisk (*p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: One-Vector System for Multiplexed CRISPR/Cas9 against Hepatitis B Virus cccDNA Utilizing High-Capacity Adenoviral Vectors

    doi: 10.1016/j.omtn.2018.05.006

    Figure Lengend Snippet: Proof of Concept of Anti-HBV Efficacy of TALENs or CRISPR/Cas9 (A) HEK293 cells were co-transfected with an HBV expression plasmid pTHBV2 and either of the complete nuclease systems. DNA was isolated after 4 days and examined for mutagenesis at target sites by T7E1 assay. Undigested and digested products were separated on an agarose gel side by side for comparison. Expected cleavage product sizes are indicated by arrowheads. “M” indicates the molecular weight marker lane. (B) Huh7 cells were co-transfected with an HBV expression plasmid pTHBV2 and TALEN or CRISPR/Cas9 nuclease systems. HBsAg concentrations in the supernatant were measured after 4 days by ELISA. Data are represented as means of S/CO (sample to control ratio) values and error bars indicate SD of three replicates. Statistically significant differences to HBV only are indicated by an asterisk (*p

    Article Snippet: Mutation Detection For the T7 endonuclease 1 (T7E1) (New England Biolabs, MA) mutation detection assay, DNA sequences that span the target sites were amplified from purified genomic DNA by PCR using standard conditions for the OneTaq 2X Master Mix with Standard Buffer (New England Biolabs, MA), with the following primer sets: S/HBV-RT, S-T7 for 5′-ttcctcttcatcctgctgct-3′ and S rev 5′-tgtaaaaggggcagcaaaac-3′; X, No. 5-forw 5′-actcctagccgcttgttttg-3′ and No.5-rev 5′-ataagggtcgatgtccatgc-3′; C, C-T7 for 5′-ctgggtgggtgttaatttgg-3′ and No.6-rev 5′-tacccgccttccatagagtg-3′; P1, P1_F 5′-gctttcactttctcgccaac-3′ and P1_R 5′-accttgggcaatatttggtg-3′; and XCp, XCp_F 5′-actctctcgtccccttctcc-3′ and XCp_R 5′-gcctgagtgcagtatggtga-3′.

    Techniques: TALENs, CRISPR, Transfection, Expressing, Plasmid Preparation, Isolation, Mutagenesis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay

    SMD is sensitive to T7 endonuclease I. Logarithmically growing apn1/2 cells were arrested in G2/M with nocodazole, treated with MMS (0.1%, 15 min) in PBS and returned to the YPDA+nocodazole medium. Cells were collected at the indicated times and processed for DNA plug preparation. The plugs were then incubated with T7 endonuclease I as described in the Materials and Methods or in buffer only (mock-treated). The SMD was estimated in the ethidium bromide stained gel by comparing the amount of DNA material in the SMD region to DNA in the small chromosomes that experienced little breakage, “SMD / Chr (I+VI)”.

    Journal: PLoS Genetics

    Article Title: Alkylation Base Damage Is Converted into Repairable Double-Strand Breaks and Complex Intermediates in G2 Cells Lacking AP Endonuclease

    doi: 10.1371/journal.pgen.1002059

    Figure Lengend Snippet: SMD is sensitive to T7 endonuclease I. Logarithmically growing apn1/2 cells were arrested in G2/M with nocodazole, treated with MMS (0.1%, 15 min) in PBS and returned to the YPDA+nocodazole medium. Cells were collected at the indicated times and processed for DNA plug preparation. The plugs were then incubated with T7 endonuclease I as described in the Materials and Methods or in buffer only (mock-treated). The SMD was estimated in the ethidium bromide stained gel by comparing the amount of DNA material in the SMD region to DNA in the small chromosomes that experienced little breakage, “SMD / Chr (I+VI)”.

    Article Snippet: Resolution of the slow moving DNA (SMD) intermediate with T7 endonuclease I DNA was digested in agarose plugs with T7 endonuclease I (New England Biolabs, Beverly, MA).

    Techniques: Incubation, Staining

    ZFN-mediated disruption of myostatin gene in primary pig fibroblasts. (A) Myostatin-targeted ZFNs used in this research. The ZFNs target sequences are indicated by the red color, each of ZFN monomer recognizes 15 bases, and five bases space between the two ZFN monomers, each zinc-finger protein recognizes three bases, the Fok I nucleases are acted as dimer to non-specific cleavage, identify, insertion and deletion. (B) Immunofluoresence staining analysis the myostatin-ZFN location. The DAPI staining nucleus (blue) and the Flag location myostain-ZFN (green). (C) Schematic overview of mismatch detection by T7 endonuclease I (T7E1). Genomic DNA was extracted from primary pig fibroblasts treated with ZFNs after 48 h of transfection, the first exon of myostatin gene that encompassing the site of ZFNs recognition was amplified by PCR, and the DNA amplicons were denaturized and annealed to form homoduplexes and heteroduplexes. The T7E1 recognizes and cleaves the mismatch DNA of heteroduplexes instead of homoduplexes, proceeding the DNA fragments analysis by agarose gel electrophoresis and the ideal result is shown. (D) T7E1 assay of ZFN cleavage in primary porcine fibroblasts. A band cleave by the T7 endonuclease I was detected at an expected size (∼325 bp), indicating the presence of heteroduplexes. ZFN, amplicons from ZFNs treated primary porcine fibroblasts; WT, amplicons from wild-type primary pig fibroblast.

    Journal: Molecules and Cells

    Article Title: Disruption of the Myostatin Gene in Porcine Primary Fibroblasts and Embryos Using Zinc-Finger Nucleases

    doi: 10.14348/molcells.2014.2209

    Figure Lengend Snippet: ZFN-mediated disruption of myostatin gene in primary pig fibroblasts. (A) Myostatin-targeted ZFNs used in this research. The ZFNs target sequences are indicated by the red color, each of ZFN monomer recognizes 15 bases, and five bases space between the two ZFN monomers, each zinc-finger protein recognizes three bases, the Fok I nucleases are acted as dimer to non-specific cleavage, identify, insertion and deletion. (B) Immunofluoresence staining analysis the myostatin-ZFN location. The DAPI staining nucleus (blue) and the Flag location myostain-ZFN (green). (C) Schematic overview of mismatch detection by T7 endonuclease I (T7E1). Genomic DNA was extracted from primary pig fibroblasts treated with ZFNs after 48 h of transfection, the first exon of myostatin gene that encompassing the site of ZFNs recognition was amplified by PCR, and the DNA amplicons were denaturized and annealed to form homoduplexes and heteroduplexes. The T7E1 recognizes and cleaves the mismatch DNA of heteroduplexes instead of homoduplexes, proceeding the DNA fragments analysis by agarose gel electrophoresis and the ideal result is shown. (D) T7E1 assay of ZFN cleavage in primary porcine fibroblasts. A band cleave by the T7 endonuclease I was detected at an expected size (∼325 bp), indicating the presence of heteroduplexes. ZFN, amplicons from ZFNs treated primary porcine fibroblasts; WT, amplicons from wild-type primary pig fibroblast.

    Article Snippet: T7 endonuclease I mutation-detection assay ZFNs-induced fibroblast and oocyte mutations were detected using a T7E1 assay (New England BioLabs, USA) following the manufacturer’s protocol and a recently reported method ( ).

    Techniques: Staining, Transfection, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    ZFN-mediated disruption of myostatin gene in porcine PA embryos. (A) T7E1 assay of ZFN cleavage in porcine PA embryos. The 425 bp PCR amplicon of myostatin gene is cleaved into ∼325 bp and ∼100 bp fragments. ZFN, the embryo mutant after ZFN coding mRNA treated; Con, the embryo treated with nonsense mRNA. (B) DNA sequencing diagram of an embryo mutant. The diagram of sequence reveals a double curve around ZFNs binding site. Arrow indicated mutant point. (C) Effect on development of PA embryos after microinjected with ZFN-coding mRNA in 1-cell. After injected with ZFN-coding mRNA in 1-cell, the developmental rate of embryos from 2-cell to blastula was shown. There was no significant difference between control and ZFN-treated group (p > 0.05).

    Journal: Molecules and Cells

    Article Title: Disruption of the Myostatin Gene in Porcine Primary Fibroblasts and Embryos Using Zinc-Finger Nucleases

    doi: 10.14348/molcells.2014.2209

    Figure Lengend Snippet: ZFN-mediated disruption of myostatin gene in porcine PA embryos. (A) T7E1 assay of ZFN cleavage in porcine PA embryos. The 425 bp PCR amplicon of myostatin gene is cleaved into ∼325 bp and ∼100 bp fragments. ZFN, the embryo mutant after ZFN coding mRNA treated; Con, the embryo treated with nonsense mRNA. (B) DNA sequencing diagram of an embryo mutant. The diagram of sequence reveals a double curve around ZFNs binding site. Arrow indicated mutant point. (C) Effect on development of PA embryos after microinjected with ZFN-coding mRNA in 1-cell. After injected with ZFN-coding mRNA in 1-cell, the developmental rate of embryos from 2-cell to blastula was shown. There was no significant difference between control and ZFN-treated group (p > 0.05).

    Article Snippet: T7 endonuclease I mutation-detection assay ZFNs-induced fibroblast and oocyte mutations were detected using a T7E1 assay (New England BioLabs, USA) following the manufacturer’s protocol and a recently reported method ( ).

    Techniques: Polymerase Chain Reaction, Amplification, Mutagenesis, DNA Sequencing, Sequencing, Binding Assay, Injection

    CRISRP-Cas9 activity reported with the T7E1 Assay and Next-Generation Sequencing. ( A ) NHEJ frequency with the T7E1 assay. Representative gel images of T7E1-treated PCR products amplified from the target sites of GFP-negative controls (−) and edited pools (+). ( B ) NHEJ events in CRISPR-Cas9 targets reported by NGS (black bars) or the T7E1 assay (grey bars). Data represent the mean of three biological replicates ±SEM. ( C ) Indel size spectrum (x-axis) and frequency (y-axis) identified by targeted NGS. The top four most prevalent reads are shown below the sgRNA sequence (5′ to 3′) with corresponding deletions (black dashes) and insertions (red letters). Red arrows identify sgRNA cut sites. Data represent the mean of three biological replicates ±SEM.

    Journal: Scientific Reports

    Article Title: A Survey of Validation Strategies for CRISPR-Cas9 Editing

    doi: 10.1038/s41598-018-19441-8

    Figure Lengend Snippet: CRISRP-Cas9 activity reported with the T7E1 Assay and Next-Generation Sequencing. ( A ) NHEJ frequency with the T7E1 assay. Representative gel images of T7E1-treated PCR products amplified from the target sites of GFP-negative controls (−) and edited pools (+). ( B ) NHEJ events in CRISPR-Cas9 targets reported by NGS (black bars) or the T7E1 assay (grey bars). Data represent the mean of three biological replicates ±SEM. ( C ) Indel size spectrum (x-axis) and frequency (y-axis) identified by targeted NGS. The top four most prevalent reads are shown below the sgRNA sequence (5′ to 3′) with corresponding deletions (black dashes) and insertions (red letters). Red arrows identify sgRNA cut sites. Data represent the mean of three biological replicates ±SEM.

    Article Snippet: The heterocomplexed PCR product (5 µL) was incubated with 5 U T7E1 enzyme (New England Bio Labs) at 37 °C for 20 min. Products from mismatch assays were electrophoresed on a Novex 10% TBE gel (Invitrogen).

    Techniques: Activity Assay, Next-Generation Sequencing, Non-Homologous End Joining, Polymerase Chain Reaction, Amplification, CRISPR, Sequencing

    Heteroduplex formation by the cDNA of de novo transcripts of TNFSF9 from HepG2 clone 9-1 Total RNA and EU-labeled RNAs were isolated from clone 9-1 HepG2 cells. cDNA was synthesized and the TNFSF9 cDNA was amplified by PCR as described in “Materials and Methods”. The PCR products were annealed and incubated in the presence or absence of T7E1 endonuclease, and the products were fractionated on 1.2% agarose gels and visualized under UV. This is one representative of three independent experiments.

    Journal: Molecules and Cells

    Article Title: Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript

    doi: 10.14348/molcells.2018.0209

    Figure Lengend Snippet: Heteroduplex formation by the cDNA of de novo transcripts of TNFSF9 from HepG2 clone 9-1 Total RNA and EU-labeled RNAs were isolated from clone 9-1 HepG2 cells. cDNA was synthesized and the TNFSF9 cDNA was amplified by PCR as described in “Materials and Methods”. The PCR products were annealed and incubated in the presence or absence of T7E1 endonuclease, and the products were fractionated on 1.2% agarose gels and visualized under UV. This is one representative of three independent experiments.

    Article Snippet: For T7 endonuclease 1 activity assay, PCR products were annealed and incubated with T7E1 (NEB, Ipswich, MA) according to the instruction manual.

    Techniques: Labeling, Isolation, Synthesized, Amplification, Polymerase Chain Reaction, Incubation

    MG132 increases the frequency of ZFN-driven mutations. After transfection of plasmids encoding ZFNs ( A : ZFN-224; B : K230), 293T cells were treated with various concentrations of MG132. Genomic DNA was isolated and ZFN-driven mutations were analyzed by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by T7E1. ( A ) ZFN-224: Because the target site lies in the center of the amplicon (780 bp), T7E1 treatment of the heteroduplexed DNA gave rise to two DNA bands with almost same size (387 bp and 389 bp), which appear as a single band. ( B ) K230: Because the target site does not lie in the center of the ampicon, T7E1 treatment of the heteroduplexed DNA gave rise to two DNA bands (493 bp and 311 bp), observed as two separate bands. The numbers at the bottom of the gel denote mutation percentages calculated by band intensities. n = 3. * P

    Journal: PLoS ONE

    Article Title: Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132

    doi: 10.1371/journal.pone.0054282

    Figure Lengend Snippet: MG132 increases the frequency of ZFN-driven mutations. After transfection of plasmids encoding ZFNs ( A : ZFN-224; B : K230), 293T cells were treated with various concentrations of MG132. Genomic DNA was isolated and ZFN-driven mutations were analyzed by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by T7E1. ( A ) ZFN-224: Because the target site lies in the center of the amplicon (780 bp), T7E1 treatment of the heteroduplexed DNA gave rise to two DNA bands with almost same size (387 bp and 389 bp), which appear as a single band. ( B ) K230: Because the target site does not lie in the center of the ampicon, T7E1 treatment of the heteroduplexed DNA gave rise to two DNA bands (493 bp and 311 bp), observed as two separate bands. The numbers at the bottom of the gel denote mutation percentages calculated by band intensities. n = 3. * P

    Article Snippet: The PCR amplicons were denatured by heating and annealed to form heteroduplex DNA, which was treated with 5 units of mismatch-sensitive T7 endonuclease 1 (New England Biolabs, Hitchin, UK) for 20 mins at 37°C and then analyzed by 2% agarose gel electrophoresis.

    Techniques: Transfection, Isolation, Amplification, Mutagenesis

    In-frame PuroR HDR donor design allows highly specific selection of mNG-STAT3 knock-in Jurkat T cells. (A) Cas9 nickase gRNA pair design targeting human STAT3 locus to induce HDR events near the start codon. (B) T7EN assay for locus-specific editing in HEK293T cells. (C) STAT3 HDR donor plasmid. Start codon and the original second codon are labeled as shown in A. (D) Strategy for puromycin selection of clonal Jurkat cells with the STAT3 locus-targeted Jurkat T cells. (E) mNeonGreen expression by clones of puromycin-resistant mNG-STAT3 Jurkat cells. (F) PCR analysis of the hSTAT3 locus in targeted mNG-STAT3 Jurkat cells. (G) Targeting efficiency in the mNG-STAT3 line.

    Journal: bioRxiv

    Article Title: Efficient Immune Cell Genome Engineering with Improved CRISPR Editing Tools

    doi: 10.1101/2020.02.13.947002

    Figure Lengend Snippet: In-frame PuroR HDR donor design allows highly specific selection of mNG-STAT3 knock-in Jurkat T cells. (A) Cas9 nickase gRNA pair design targeting human STAT3 locus to induce HDR events near the start codon. (B) T7EN assay for locus-specific editing in HEK293T cells. (C) STAT3 HDR donor plasmid. Start codon and the original second codon are labeled as shown in A. (D) Strategy for puromycin selection of clonal Jurkat cells with the STAT3 locus-targeted Jurkat T cells. (E) mNeonGreen expression by clones of puromycin-resistant mNG-STAT3 Jurkat cells. (F) PCR analysis of the hSTAT3 locus in targeted mNG-STAT3 Jurkat cells. (G) Targeting efficiency in the mNG-STAT3 line.

    Article Snippet: For the T7EN assay, equal amounts of genomic PCR products were used within each comparison group for the hybridization reaction in a thermocycler programmed to 95°C for 10 minutes, -2°C per second ramp to 85°C, -0.1°C per second ramp to 25°C, and then held at 4°C until a subsequent T7 Endonuclease I (NEB) treatment at 37°C for 30 to 90 minutes.

    Techniques: Selection, Knock-In, Plasmid Preparation, Labeling, Expressing, Clone Assay, Polymerase Chain Reaction

    CRE-loxP excision strategy minimizes genomic footprint of the PuroR selection cassette and allows for multiple rounds of genome editing. (A) Cas9 nickase gRNA pair design targeting the human BCL10 locus. (B) T7EN assay for locus-specific editing in HEK293T cells. (C) Double gRNAs and Cas9-D10A nickase expression plasmid derived from the PX462 backbone. (D) BCL10 HDR donor plasmid design. (E) Strategy for creating puromycin-selected Jurkat T cells with post-selection excision of the Puro-T2A cassette. (F)(G) mNeonGreen expression by puromycin-selected mNG-BCL10 knock-in Jurkat line with or without NIL-CRE treatment. (H) Modified STAT3 HDR donor plasmid. (I) mNeonGreen and mScarlet co-expression by puromycin-selected mScarlet-STAT3/mNG-BCL10 double knock-in Jurkat line.

    Journal: bioRxiv

    Article Title: Efficient Immune Cell Genome Engineering with Improved CRISPR Editing Tools

    doi: 10.1101/2020.02.13.947002

    Figure Lengend Snippet: CRE-loxP excision strategy minimizes genomic footprint of the PuroR selection cassette and allows for multiple rounds of genome editing. (A) Cas9 nickase gRNA pair design targeting the human BCL10 locus. (B) T7EN assay for locus-specific editing in HEK293T cells. (C) Double gRNAs and Cas9-D10A nickase expression plasmid derived from the PX462 backbone. (D) BCL10 HDR donor plasmid design. (E) Strategy for creating puromycin-selected Jurkat T cells with post-selection excision of the Puro-T2A cassette. (F)(G) mNeonGreen expression by puromycin-selected mNG-BCL10 knock-in Jurkat line with or without NIL-CRE treatment. (H) Modified STAT3 HDR donor plasmid. (I) mNeonGreen and mScarlet co-expression by puromycin-selected mScarlet-STAT3/mNG-BCL10 double knock-in Jurkat line.

    Article Snippet: For the T7EN assay, equal amounts of genomic PCR products were used within each comparison group for the hybridization reaction in a thermocycler programmed to 95°C for 10 minutes, -2°C per second ramp to 85°C, -0.1°C per second ramp to 25°C, and then held at 4°C until a subsequent T7 Endonuclease I (NEB) treatment at 37°C for 30 to 90 minutes.

    Techniques: Selection, Expressing, Plasmid Preparation, Derivative Assay, Knock-In, Modification

    Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p

    Journal: Frontiers in Immunology

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

    doi: 10.3389/fimmu.2018.01680

    Figure Lengend Snippet: Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p

    Article Snippet: Treatment with DNase I did not modify rates of weight gain (or spleen weight/body weight ratios) (Figures S4A,B in Supplementary Material).

    Techniques: Mouse Assay, Immunofluorescence, Microscopy, Staining

    Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

    doi: 10.3389/fimmu.2018.01680

    Figure Lengend Snippet: Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p

    Article Snippet: Treatment with DNase I did not modify rates of weight gain (or spleen weight/body weight ratios) (Figures S4A,B in Supplementary Material).

    Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Staining, Immunofluorescence, Microscopy

    hSWI/SNF remodeling of nucleosomes containing branched and nicked DNAs. (A) Analysis of hSWI/SNF remodeling of nucleosomes reconstituted with native, flap, hairpin, and nick templates by DNase I digestion. In each panel, lanes 1 show G-specific reaction markers of the native 5S DNA fragment, lanes 2 show DNase I digestion pattern of naked templates, lanes 3 are nucleosomes prior to DNase I digestion, lanes 4 show the DNase I cleavage pattern of nucleosomes incubated with hSWI/SNF without ATP, and lanes 5 show the DNase I cleavage patterns of nucleosomes incubated with hSWI/SNF and ATP for 15 min. (B) Effects of branched DNA structures on remodeling as determined by restriction enzyme accessibility assay. Glycerol-gradient-purified native, nicked, hairpin, and flap nucleosomes were incubated for the indicated times with hSWI/SNF in the presence or absence of ATP (open and solid symbols, respectively) and subjected to Eco RV digestion over a 45-min time course. The percent nucleosomes remaining uncut is plotted versus time of Eco RV digestion for native (diamonds), hairpin (squares), flap (triangles), and nicked (circles) nucleosomes.

    Journal: Molecular and Cellular Biology

    Article Title: hSWI/SNF-Catalyzed Nucleosome Sliding Does Not Occur Solely via a Twist-Diffusion Mechanism

    doi: 10.1128/MCB.22.21.7484-7490.2002

    Figure Lengend Snippet: hSWI/SNF remodeling of nucleosomes containing branched and nicked DNAs. (A) Analysis of hSWI/SNF remodeling of nucleosomes reconstituted with native, flap, hairpin, and nick templates by DNase I digestion. In each panel, lanes 1 show G-specific reaction markers of the native 5S DNA fragment, lanes 2 show DNase I digestion pattern of naked templates, lanes 3 are nucleosomes prior to DNase I digestion, lanes 4 show the DNase I cleavage pattern of nucleosomes incubated with hSWI/SNF without ATP, and lanes 5 show the DNase I cleavage patterns of nucleosomes incubated with hSWI/SNF and ATP for 15 min. (B) Effects of branched DNA structures on remodeling as determined by restriction enzyme accessibility assay. Glycerol-gradient-purified native, nicked, hairpin, and flap nucleosomes were incubated for the indicated times with hSWI/SNF in the presence or absence of ATP (open and solid symbols, respectively) and subjected to Eco RV digestion over a 45-min time course. The percent nucleosomes remaining uncut is plotted versus time of Eco RV digestion for native (diamonds), hairpin (squares), flap (triangles), and nicked (circles) nucleosomes.

    Article Snippet: For the DNase I assay, the bottom 215-mer strand was 3′ end labeled with the Klenow fragment (New England Biolabs) prior to the annealing reaction.

    Techniques: Incubation, Purification