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  • 98
    Bio-Rad electroporation cuvettes bio rad
    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to <t>electroporation</t> number/colony number.
    Electroporation Cuvettes Bio Rad, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 98/100, based on 309 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad sterile bio rad genepulser electroporation cuvette
    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to <t>electroporation</t> number/colony number.
    Sterile Bio Rad Genepulser Electroporation Cuvette, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad pre cooled bio rad genepulser electroporation cuvette
    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to <t>electroporation</t> number/colony number.
    Pre Cooled Bio Rad Genepulser Electroporation Cuvette, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad electrode gap bio rad electroporation cuvette
    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to <t>electroporation</t> number/colony number.
    Electrode Gap Bio Rad Electroporation Cuvette, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad bio rad cuvette
    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to <t>electroporation</t> number/colony number.
    Bio Rad Cuvette, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 98/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Bio-Rad 0 4 cm path length sterile bio rad electroporation cuvette
    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to <t>electroporation</t> number/colony number.
    0 4 Cm Path Length Sterile Bio Rad Electroporation Cuvette, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad electroporation
    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to <t>electroporation</t> number/colony number.
    Electroporation, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 14452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad electroporation cuvets
    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to <t>electroporation</t> number/colony number.
    Electroporation Cuvets, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad bio rad gene pulser cuvette
    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to <t>electroporation</t> number/colony number.
    Bio Rad Gene Pulser Cuvette, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad micropulser electroporation cuvette
    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to <t>electroporation</t> number/colony number.
    Micropulser Electroporation Cuvette, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad generic electroporation cuvettes
    Using SCIPay to insert a chimeric antigen receptor (CAR) into the T cell receptor alpha subunit (TRAC) gene. (A) Overview of SCIPay designed to insert an anti-EGFR CAR with BFP into the human TRAC gene. (B) Outline of cell engineering strategy. After <t>electroporation,</t> successfully transfected cells were sorted twice to enrich BFP-positive cells. (C) BFP and TCR/TRAC expression on double-sorted population (B1). (D) Surface CD69 expression during co-culture between the indicated cell population and SKOV3 or MCF7 cells. Results in (D) represent means +/- SEM of 3 independent experiments.
    Generic Electroporation Cuvettes, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad mmgap electroporation cuvettes
    Using SCIPay to insert a chimeric antigen receptor (CAR) into the T cell receptor alpha subunit (TRAC) gene. (A) Overview of SCIPay designed to insert an anti-EGFR CAR with BFP into the human TRAC gene. (B) Outline of cell engineering strategy. After <t>electroporation,</t> successfully transfected cells were sorted twice to enrich BFP-positive cells. (C) BFP and TCR/TRAC expression on double-sorted population (B1). (D) Surface CD69 expression during co-culture between the indicated cell population and SKOV3 or MCF7 cells. Results in (D) represent means +/- SEM of 3 independent experiments.
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    Bio-Rad electroporation chamber
    Using SCIPay to insert a chimeric antigen receptor (CAR) into the T cell receptor alpha subunit (TRAC) gene. (A) Overview of SCIPay designed to insert an anti-EGFR CAR with BFP into the human TRAC gene. (B) Outline of cell engineering strategy. After <t>electroporation,</t> successfully transfected cells were sorted twice to enrich BFP-positive cells. (C) BFP and TCR/TRAC expression on double-sorted population (B1). (D) Surface CD69 expression during co-culture between the indicated cell population and SKOV3 or MCF7 cells. Results in (D) represent means +/- SEM of 3 independent experiments.
    Electroporation Chamber, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to electroporation number/colony number.

    Journal: Molecular Plant Pathology

    Article Title: Pectate lyase affects pathogenicity in natural isolates of Colletotrichum coccodes and in pelA gene‐disrupted and gene‐overexpressing mutant lines

    doi: 10.1111/j.1364-3703.2011.00740.x

    Figure Lengend Snippet: Targeted gene disruption of CcpelA . (A–C) Schematic diagram of the strategy used for targeted gene disruption of CcpelA encoding pectate lyase (PL). (A) CcpelA in wild‐type (WT) genome. (B) Linearized pGH1‐1240PL plasmid containing CcpelA sequence. (C) Genome organization of CcpelA disrupted via homologous recombination. Box colours: dark and light grey, CcpelA gene sequence (sequence marked in dark grey was included in the disruption construct); black, sequences derived from pGEM vector; white shading, hygromycin resistance gene; white, other vector sequences. Arrows indicate the positions and orientations of the polymerase chain reaction (PCR) primers used for verification of the transformants: 1, For‐new‐PL4; 2, Rev‐new‐PL4; 3, For‐pGEM; 4, Rev‐pGEM. (D–F) PCR analysis of CcpelA gene‐disrupted transformants. Genomic DNA isolated from wild‐type (WT) isolates US‐41 and Si‐72, CcpelA gene‐disrupted transformants US‐41/7.1 and Si‐72/2.1, and representatives of ectopic transformants US‐41/5.1 and Si‐72/5.1 were analysed using the following primers sets: (D) For‐new‐PL4 and Rev‐new‐PL4 (1 and 2 in A and C); (E) For‐new‐PL4 and For‐pGEM (1 and 3 in C); (F) Rev‐new‐PL4 and Rev‐pGEM (2 and 4 in C). All PCR products were run on a 1% agarose gel and stained with ethidium bromide. Mutants were numbered according to electroporation number/colony number.

    Article Snippet: Aliquots (0.1 mL) were placed into cold 0.2‐cm electroporation cuvettes (Bio‐Rad) and linearized DNA plasmid (5–10 µg) was added.

    Techniques: Plasmid Preparation, Sequencing, Homologous Recombination, Construct, Derivative Assay, Polymerase Chain Reaction, Isolation, Agarose Gel Electrophoresis, Staining, Electroporation

    Effect of Ref-1 on transactivation activity of the Δ71-BSAP promoter. ( A ) Plasmids were transfected by the calcium phosphate method in HeLa cells at the concentrations indicated in Materials and Methods. Forty-eight hours after transfection, cells were harvested and CAT and Luc activities were measured in nuclear extracts. Bars indicate the means ± SD of at least three independent experiments. ( B ) Plasmids were transfected by electroporation into NS-1 cells at the concentrations indicated in Materials and Methods. Twenty-four hours after transfection, cells were harvested and CAT and Luc activities were measured in nuclear extracts. Bars indicate the means ± SD of at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: An 'environment to nucleus' signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation

    doi:

    Figure Lengend Snippet: Effect of Ref-1 on transactivation activity of the Δ71-BSAP promoter. ( A ) Plasmids were transfected by the calcium phosphate method in HeLa cells at the concentrations indicated in Materials and Methods. Forty-eight hours after transfection, cells were harvested and CAT and Luc activities were measured in nuclear extracts. Bars indicate the means ± SD of at least three independent experiments. ( B ) Plasmids were transfected by electroporation into NS-1 cells at the concentrations indicated in Materials and Methods. Twenty-four hours after transfection, cells were harvested and CAT and Luc activities were measured in nuclear extracts. Bars indicate the means ± SD of at least three independent experiments.

    Article Snippet: Aliquots of 1.5 × 107 cells were resuspended in 350 µl of RPMI in a Bio-Rad electroporation cuvette (0.4 cm gap) and pulsed once with 340 V and 960 µF.

    Techniques: Activity Assay, Transfection, Electroporation

    14-3-3τ is required for the expression of Beclin 1. (A) HEK293 and U2OS cells were transiently transfected with pSUPER-si14-3-3τ [15] or pSUPER expressing a scrambled sequence (siScr) [37] . Forty-four hr later, cells were harvested and RNA was extracted. Real-time RT-PCR analysis was performed using primers specific for Beclin 1, 14-3-3τ and GAPDH. The levels of Beclin 1 and 14-3-3τ were normalized to GAPDH levels and are expressed relative to the expression of the gene in the siScr control. The data shown represent the means ± standard deviations of triplicate samples. The p values are based on a paired two-tailed t test. (B) We established stable U2OS cell lines expressing an inducible siRNA against 14-3-3τ or a control GFP sequence under the control of tetracycline operon in pSUPERIOR.puro vector (OligoEngine). 14-3-3τ siRNA or a GFP siRNA was induced by doxycycline (DOX) (1 µg/ml), and cells were harvested for Western blot analysis using anti-Beclin 1, anti-14-3-3τ or anti-GAPDH antibody. (C) HEK293 cells were transiently transfected by three different pSUPER-14-3-3τ siRNA constructs (20 µg) by a calcium phosphate method. The cell lysates were harvested 48 hr later and analyzed by Western blot analysis. (D) HCT116 cells were transfected with 20 µg pSUPER-scrambled siRNA (siS) or si14-3-3τ (si14) along with a pcDNA3 empty vector or a vector expressing siRNA-resistant 14-3-3τ (res) by electroporation. The cell lysates were harvested 44 hr later and subject to Western blot analysis as indicated. (E) MCF7 cells were infected with a recombinant adenovirus expressing a control siGFP or si14-3-3τ (si14) at MOI of 100. Forty-two hr later, cells were harvested for Western blot analysis as indicated.

    Journal: PLoS ONE

    Article Title: 14-3-3? Regulates Beclin 1 and Is Required for Autophagy

    doi: 10.1371/journal.pone.0010409

    Figure Lengend Snippet: 14-3-3τ is required for the expression of Beclin 1. (A) HEK293 and U2OS cells were transiently transfected with pSUPER-si14-3-3τ [15] or pSUPER expressing a scrambled sequence (siScr) [37] . Forty-four hr later, cells were harvested and RNA was extracted. Real-time RT-PCR analysis was performed using primers specific for Beclin 1, 14-3-3τ and GAPDH. The levels of Beclin 1 and 14-3-3τ were normalized to GAPDH levels and are expressed relative to the expression of the gene in the siScr control. The data shown represent the means ± standard deviations of triplicate samples. The p values are based on a paired two-tailed t test. (B) We established stable U2OS cell lines expressing an inducible siRNA against 14-3-3τ or a control GFP sequence under the control of tetracycline operon in pSUPERIOR.puro vector (OligoEngine). 14-3-3τ siRNA or a GFP siRNA was induced by doxycycline (DOX) (1 µg/ml), and cells were harvested for Western blot analysis using anti-Beclin 1, anti-14-3-3τ or anti-GAPDH antibody. (C) HEK293 cells were transiently transfected by three different pSUPER-14-3-3τ siRNA constructs (20 µg) by a calcium phosphate method. The cell lysates were harvested 48 hr later and analyzed by Western blot analysis. (D) HCT116 cells were transfected with 20 µg pSUPER-scrambled siRNA (siS) or si14-3-3τ (si14) along with a pcDNA3 empty vector or a vector expressing siRNA-resistant 14-3-3τ (res) by electroporation. The cell lysates were harvested 44 hr later and subject to Western blot analysis as indicated. (E) MCF7 cells were infected with a recombinant adenovirus expressing a control siGFP or si14-3-3τ (si14) at MOI of 100. Forty-two hr later, cells were harvested for Western blot analysis as indicated.

    Article Snippet: The Gene Pulser Xcell electroporation conditions for HCT116 cells are: Voltage 155 V, Capacitance 1000 µF, Resistance ∝Ω, 20 µg DNA in 200 µl tranfection medium (McCoy's 5A without supplement) within 2 mm Bio-Rad electroporation cuvettes.

    Techniques: Expressing, Transfection, Sequencing, Quantitative RT-PCR, Two Tailed Test, Plasmid Preparation, Western Blot, Construct, Electroporation, Infection, Recombinant

    Functional analysis of HmTSP by siRNA knockdown. ( a ) ISH analysis of HmTSP expression in control GFP -transgenic animals electroporated with GFP siRNA. ( b ) ISH analysis showing reduced HmTSP expression in animals electroporated with HmTSP siRNA. Bars = 100 µm. ( c , d ) Demonstration of effective siRNA knockdown for GFP in transgenic hydra with ectodermal GFP expression. ( c ) Untreated control animal. D. Representative animal of the same transgenic strain as in C 8 days after electroporation with GFP siRNA. Bars = 500 µm. ( e– e’) Treatment with combined siGFP and siHmTSP does not induce morphological changes in steady state polyps. ( f , g ) Treatment with ALP of siTSP electroporated animals resulted in a dramatic increase of ectopic tentacles compared to the siGFP-treated control group. f’ and g’ show the reduced GFP expression in the respective animals. Each panel representative of at least 10 hydras examined. ( h ) Quantification of ALP-induced ectopic tentacles in animals electroporated with siGFP or siGFP and siTSP. Animals electroporated with the respective siRNAs without subsequent ALP treatment served as controls. ALP treatment was performed 8 days after electroporation and the numbers of tentacles/animal in each group were counted 5 days after ALP treatment. Animals in d and e were recorded at the same time point after electroporation as animals in f and g. Animals (n) in each group were: siGFP = 67; siGFP/siTSP = 67; siGFP/ALP = 71; siGFP/siTSP/ALP = 71. ( i ) Effect of Wnt3 depletion on ectopic tentacle formation: Animals were electroporated with siRNAs specific for GFP or directed against Wnt3 and GFP followed by ALP- or control treatment as indicated. Ectopic tentacle formation was analyzed as described above ( h ). Number of polyps analyzed in each group was: siGFP: 74, siGFP/siWnt3: 76, siGFP/ALP: 79, and siGFP/siWnt3/ALP: 81. ( h and i show the results from three independent experiments. Each data point represents a single hydra, bars indicate the mean ± S.E.M. ***P value

    Journal: Scientific Reports

    Article Title: Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction

    doi: 10.1038/s41598-018-30035-2

    Figure Lengend Snippet: Functional analysis of HmTSP by siRNA knockdown. ( a ) ISH analysis of HmTSP expression in control GFP -transgenic animals electroporated with GFP siRNA. ( b ) ISH analysis showing reduced HmTSP expression in animals electroporated with HmTSP siRNA. Bars = 100 µm. ( c , d ) Demonstration of effective siRNA knockdown for GFP in transgenic hydra with ectodermal GFP expression. ( c ) Untreated control animal. D. Representative animal of the same transgenic strain as in C 8 days after electroporation with GFP siRNA. Bars = 500 µm. ( e– e’) Treatment with combined siGFP and siHmTSP does not induce morphological changes in steady state polyps. ( f , g ) Treatment with ALP of siTSP electroporated animals resulted in a dramatic increase of ectopic tentacles compared to the siGFP-treated control group. f’ and g’ show the reduced GFP expression in the respective animals. Each panel representative of at least 10 hydras examined. ( h ) Quantification of ALP-induced ectopic tentacles in animals electroporated with siGFP or siGFP and siTSP. Animals electroporated with the respective siRNAs without subsequent ALP treatment served as controls. ALP treatment was performed 8 days after electroporation and the numbers of tentacles/animal in each group were counted 5 days after ALP treatment. Animals in d and e were recorded at the same time point after electroporation as animals in f and g. Animals (n) in each group were: siGFP = 67; siGFP/siTSP = 67; siGFP/ALP = 71; siGFP/siTSP/ALP = 71. ( i ) Effect of Wnt3 depletion on ectopic tentacle formation: Animals were electroporated with siRNAs specific for GFP or directed against Wnt3 and GFP followed by ALP- or control treatment as indicated. Ectopic tentacle formation was analyzed as described above ( h ). Number of polyps analyzed in each group was: siGFP: 74, siGFP/siWnt3: 76, siGFP/ALP: 79, and siGFP/siWnt3/ALP: 81. ( h and i show the results from three independent experiments. Each data point represents a single hydra, bars indicate the mean ± S.E.M. ***P value

    Article Snippet: For each reaction, 20 animals were transferred to electroporation cuvettes (4 mm gap, Bio-Rad) and excess liquid was removed.

    Techniques: Functional Assay, In Situ Hybridization, Expressing, Transgenic Assay, Electroporation, ALP Assay

    Electric field enhancement by CNT in electroporation. (a) Sketch of electroporation with CNT-free (left) and CNT associated cells (right). Table insert: theoretical electric field ( E ) enhancement over the applied field ( E 0 ) by a nanotube of 1 μ m long. d denotes the distance to nanotube tip. a 1 and a 2 are the aspect ratios (i.e. diameter/length) of nanotubes, and equal to 0.1 and 0.5 respectively. (b) Electroporation time constants versus the voltages. A series of time constants of the CNT associated Bal17 cells were obtained under various voltages. They were compared to the time constant of regular Bal17 cells at 230 V. (c) Improvement of EGFP transfection by CNT mediated electroporation in Bal17 cells. The gates of EGFP positive cells were adjusted based on the Neg Contl and Pos Contl . The mean values of EGFP levels (left axis) and the percentages (right axis) of gated viable cells are shown. Neg Contl : negative control, cells speared and electroporated without plasmid; Pos Contl : positive control, cells without spearing and electroporated at 230 V with EGFP plasmid; 180, 200 and 220 V: CNT associated cells and electroporated with EGFP plasmids at corresponding voltages.

    Journal: Nanotechnology

    Article Title: Interaction between carbon nanotubes and mammalian cells: characterization by flow cytometry and application

    doi: 10.1088/0957-4484/19/34/345102

    Figure Lengend Snippet: Electric field enhancement by CNT in electroporation. (a) Sketch of electroporation with CNT-free (left) and CNT associated cells (right). Table insert: theoretical electric field ( E ) enhancement over the applied field ( E 0 ) by a nanotube of 1 μ m long. d denotes the distance to nanotube tip. a 1 and a 2 are the aspect ratios (i.e. diameter/length) of nanotubes, and equal to 0.1 and 0.5 respectively. (b) Electroporation time constants versus the voltages. A series of time constants of the CNT associated Bal17 cells were obtained under various voltages. They were compared to the time constant of regular Bal17 cells at 230 V. (c) Improvement of EGFP transfection by CNT mediated electroporation in Bal17 cells. The gates of EGFP positive cells were adjusted based on the Neg Contl and Pos Contl . The mean values of EGFP levels (left axis) and the percentages (right axis) of gated viable cells are shown. Neg Contl : negative control, cells speared and electroporated without plasmid; Pos Contl : positive control, cells without spearing and electroporated at 230 V with EGFP plasmid; 180, 200 and 220 V: CNT associated cells and electroporated with EGFP plasmids at corresponding voltages.

    Article Snippet: Each electroporation cuvette (cooled on ice) was loaded with 3 × 106 regular or CNT associated Bal17 cells in suspension adjusted to 200 μ l. A Gene Pulser electroporation system (BIO-RAD, Hercules, CA) was High Cap, 960 μ F. Voltages were selected from 100 to 250 V. For the transfection experiments, pEGFP plasmid DNA (Clontech, Mountain View, CA) was supplemented to the cell suspensions in the cuvettes.

    Techniques: Electroporation, Transfection, Negative Control, Plasmid Preparation, Positive Control

    Overview of CRISPR-EZ technology and workflow. (a) An illustration of the most successful CRISPR-EZ editing strategies. A single sgRNA can be used to create a small indel via NHEJ repair or in conjunction with a ssODNs to create a precision mutation or a small insertion by HDR. Multiple sgRNAs can be used to engineer a genomic deletion by NHEJ repair. Design sgRNAs, HDR donor oligos, and editing validation assays prior to CRISPR-EZ experiments. (b) A graphic overview of CRISPR-EZ workflow. Day 1–3: ~4 week-old females are superovulated, first by PMSG injection, 46–48 hours later by hCG injection, before being housed with stud males for breeding. In parallel, sgRNAs are in vitro transcribed and purified. Day 4: Pronuclear stage embryos are collected and processed for electroporation, while Cas9/sgRNA complexes are assembled in vitro . Embryos (harvested at 0.5 dpc), Cas9/sgRNA RNPs, and optional ssODNs are combined in an electroporation cuvette and subjected to a series of electrical pulses. We recommend ex vivo validation of sgRNA editing efficiency in cultured morulae or blastocysts before generating edited mice. With a validated sgRNA design, electroporated embryos can be transferred to the oviduct of 0.5 dpc, pseudopregnant mothers to generate genetically engineered mice, which are then genotyped to confirm editing efficiency.

    Journal: Nature protocols

    Article Title: Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology

    doi: 10.1038/nprot.2018.012

    Figure Lengend Snippet: Overview of CRISPR-EZ technology and workflow. (a) An illustration of the most successful CRISPR-EZ editing strategies. A single sgRNA can be used to create a small indel via NHEJ repair or in conjunction with a ssODNs to create a precision mutation or a small insertion by HDR. Multiple sgRNAs can be used to engineer a genomic deletion by NHEJ repair. Design sgRNAs, HDR donor oligos, and editing validation assays prior to CRISPR-EZ experiments. (b) A graphic overview of CRISPR-EZ workflow. Day 1–3: ~4 week-old females are superovulated, first by PMSG injection, 46–48 hours later by hCG injection, before being housed with stud males for breeding. In parallel, sgRNAs are in vitro transcribed and purified. Day 4: Pronuclear stage embryos are collected and processed for electroporation, while Cas9/sgRNA complexes are assembled in vitro . Embryos (harvested at 0.5 dpc), Cas9/sgRNA RNPs, and optional ssODNs are combined in an electroporation cuvette and subjected to a series of electrical pulses. We recommend ex vivo validation of sgRNA editing efficiency in cultured morulae or blastocysts before generating edited mice. With a validated sgRNA design, electroporated embryos can be transferred to the oviduct of 0.5 dpc, pseudopregnant mothers to generate genetically engineered mice, which are then genotyped to confirm editing efficiency.

    Article Snippet: Transfer the entire 20 µl embryo/RNP mixture to a 0.1 cm gap electroporation cuvette, avoid making bubbles, and electroporate embryos in a Biorad Gene Pulser XCell apparatus with a square wave protocol and the following parameters:

    Techniques: CRISPR, Non-Homologous End Joining, Mutagenesis, Injection, In Vitro, Purification, Electroporation, Ex Vivo, Cell Culture, Mouse Assay

    Diagram illustrating a cloning-free strategy for sgRNA synthesis. The sequences and purpose of each synthesized oligo are diagrammed to show how they function in this cloning free strategy. The components include a pair of PCR primers (black: IVT-FWD and IVT-REV), a common reverse template oligo (blue/red: IVT-Scaffold-Long), and an oligo containing a 5’ T7 promoter and a unique sgRNA sequence (green/black/blue: IVT-VAR-sgRNA). The DNA template for sgRNA synthesis is generated by a PCR reaction. The product of this reaction is a single 127 bp amplicon which should be confirmed by gel electrophoresis prior to continuing. Shown on the right is a representative DNA electrophoresis image of the PCR reaction. Subsequently, sgRNAs are synthesized by T7 in vitro transcription (IVT) and purified. Before moving forward, the quality and quantity of the newly synthesized sgRNA can be determined by submitting the sample for BioAnalyzer testing. A representative bioanalyzer trace of the IVT products is shown to the right. Recombinant Cas9 protein, purified sgRNA(s), and ssODN (optional) are assembled into RNPs in vitro by combining the components with a stabilizing buffer at 37°C for 10 minutes. The active RNP Complex is now ready for electroporation.

    Journal: Nature protocols

    Article Title: Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology

    doi: 10.1038/nprot.2018.012

    Figure Lengend Snippet: Diagram illustrating a cloning-free strategy for sgRNA synthesis. The sequences and purpose of each synthesized oligo are diagrammed to show how they function in this cloning free strategy. The components include a pair of PCR primers (black: IVT-FWD and IVT-REV), a common reverse template oligo (blue/red: IVT-Scaffold-Long), and an oligo containing a 5’ T7 promoter and a unique sgRNA sequence (green/black/blue: IVT-VAR-sgRNA). The DNA template for sgRNA synthesis is generated by a PCR reaction. The product of this reaction is a single 127 bp amplicon which should be confirmed by gel electrophoresis prior to continuing. Shown on the right is a representative DNA electrophoresis image of the PCR reaction. Subsequently, sgRNAs are synthesized by T7 in vitro transcription (IVT) and purified. Before moving forward, the quality and quantity of the newly synthesized sgRNA can be determined by submitting the sample for BioAnalyzer testing. A representative bioanalyzer trace of the IVT products is shown to the right. Recombinant Cas9 protein, purified sgRNA(s), and ssODN (optional) are assembled into RNPs in vitro by combining the components with a stabilizing buffer at 37°C for 10 minutes. The active RNP Complex is now ready for electroporation.

    Article Snippet: Transfer the entire 20 µl embryo/RNP mixture to a 0.1 cm gap electroporation cuvette, avoid making bubbles, and electroporate embryos in a Biorad Gene Pulser XCell apparatus with a square wave protocol and the following parameters:

    Techniques: Clone Assay, Synthesized, Polymerase Chain Reaction, Sequencing, Generated, Amplification, Nucleic Acid Electrophoresis, In Vitro, Purification, Recombinant, Electroporation

    Comparing editing efficiency and viability of CRISPR-EZ and microinjection. (a) A schematic diagram illustrates the strategy to employ two pairs of sgRNAs to mediate the deletion of Sh3rf2 exon 5. (b,c) QIAxcel fragment analysis images show PCR genotyping results of all mice generated through microinjection ( b ) and CRISPR-EZ ( c ) in C57BL/6N mice, demonstrating a greater editing efficiency in CRISPR-EZ experiments. (NTC= non-template control, WT=wildtype) (d) Deletion efficiency of Sh3rf2 exon 5 is shown for a side-by-side comparison between CRISPR-EZ and microinjection. (e) In a high throughput pipeline, up to two pairs of sgRNAs (4 sgRNAs) were designed to mediate exon deletion in C57BL/6N mice. Average editing efficiency is calculated across 21 CRISPR-EZ experiments or 27 microinjection experiments. CRISPR-EZ outperformed microinjection in editing efficiency (left), generating an average of 3 founder animals when starting with ~60 embryos for electroporation (right). ( f) In a direct comparison, gene knockout experiments were carried out in C57BL/6N mice using either CRISPR-EZ (n=9) or microinjection (n=9), using identical sgRNA designs. CRISPR-EZ outperformed microinjection in editing efficiency (left), generating an average of 4 founder animals when starting with ~60 embryos for electroporation. (g) No obvious differences are observed for animal viability (left) or litter size (right) between CRISPR-EZ and microinjection. Data are means ± SD across all genes. ** P

    Journal: Nature protocols

    Article Title: Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology

    doi: 10.1038/nprot.2018.012

    Figure Lengend Snippet: Comparing editing efficiency and viability of CRISPR-EZ and microinjection. (a) A schematic diagram illustrates the strategy to employ two pairs of sgRNAs to mediate the deletion of Sh3rf2 exon 5. (b,c) QIAxcel fragment analysis images show PCR genotyping results of all mice generated through microinjection ( b ) and CRISPR-EZ ( c ) in C57BL/6N mice, demonstrating a greater editing efficiency in CRISPR-EZ experiments. (NTC= non-template control, WT=wildtype) (d) Deletion efficiency of Sh3rf2 exon 5 is shown for a side-by-side comparison between CRISPR-EZ and microinjection. (e) In a high throughput pipeline, up to two pairs of sgRNAs (4 sgRNAs) were designed to mediate exon deletion in C57BL/6N mice. Average editing efficiency is calculated across 21 CRISPR-EZ experiments or 27 microinjection experiments. CRISPR-EZ outperformed microinjection in editing efficiency (left), generating an average of 3 founder animals when starting with ~60 embryos for electroporation (right). ( f) In a direct comparison, gene knockout experiments were carried out in C57BL/6N mice using either CRISPR-EZ (n=9) or microinjection (n=9), using identical sgRNA designs. CRISPR-EZ outperformed microinjection in editing efficiency (left), generating an average of 4 founder animals when starting with ~60 embryos for electroporation. (g) No obvious differences are observed for animal viability (left) or litter size (right) between CRISPR-EZ and microinjection. Data are means ± SD across all genes. ** P

    Article Snippet: Transfer the entire 20 µl embryo/RNP mixture to a 0.1 cm gap electroporation cuvette, avoid making bubbles, and electroporate embryos in a Biorad Gene Pulser XCell apparatus with a square wave protocol and the following parameters:

    Techniques: CRISPR, Polymerase Chain Reaction, Mouse Assay, Generated, High Throughput Screening Assay, Electroporation, Gene Knockout

    Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability. (a) A diagram illustrates the NHEJ and HDR editing strategies for exon 1 of the Tyr gene. A successful NHEJ editing ablates a HinfI site and disrupts T yr gene function. A successful HDR editing replaces the HinfI site with an EcoRI site, introducing a frameshift mutation that abolishes Tyr gene function. (b) Representative RFLP results of Tyr edited mice indicate successful NHEJ editing (top) and HDR editing (bottom). (c) Since bi-allelic Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of Tyr editing that disrupts the genes function. Coat color (left) and viability (right) of C57B/6J edited mice generated from 2, 4, 6 or 8 pulse CRISPR-EZ conditions. Viability is defined as the percentage of live animals born out of total embryos transferred. The 6-pulse condition maximizes editing efficiency while minimally impacting pup viability. (d) Comparison of editing efficiency between C57B/6J and C57B/6N mouse strain using 2 or 6-pulse electroporation conditions. The 6-pulse CRISPR-EZ condition is equally effective in both strains. (e-i) Representative images are shown for the coat color of edited mice from experiments shown in (b-d). All animal procedures were approved by the Institutional Animal Care and Use Committee of UC Davis.

    Journal: Nature protocols

    Article Title: Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology

    doi: 10.1038/nprot.2018.012

    Figure Lengend Snippet: Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability. (a) A diagram illustrates the NHEJ and HDR editing strategies for exon 1 of the Tyr gene. A successful NHEJ editing ablates a HinfI site and disrupts T yr gene function. A successful HDR editing replaces the HinfI site with an EcoRI site, introducing a frameshift mutation that abolishes Tyr gene function. (b) Representative RFLP results of Tyr edited mice indicate successful NHEJ editing (top) and HDR editing (bottom). (c) Since bi-allelic Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of Tyr editing that disrupts the genes function. Coat color (left) and viability (right) of C57B/6J edited mice generated from 2, 4, 6 or 8 pulse CRISPR-EZ conditions. Viability is defined as the percentage of live animals born out of total embryos transferred. The 6-pulse condition maximizes editing efficiency while minimally impacting pup viability. (d) Comparison of editing efficiency between C57B/6J and C57B/6N mouse strain using 2 or 6-pulse electroporation conditions. The 6-pulse CRISPR-EZ condition is equally effective in both strains. (e-i) Representative images are shown for the coat color of edited mice from experiments shown in (b-d). All animal procedures were approved by the Institutional Animal Care and Use Committee of UC Davis.

    Article Snippet: Transfer the entire 20 µl embryo/RNP mixture to a 0.1 cm gap electroporation cuvette, avoid making bubbles, and electroporate embryos in a Biorad Gene Pulser XCell apparatus with a square wave protocol and the following parameters:

    Techniques: CRISPR, Non-Homologous End Joining, Mutagenesis, Mouse Assay, Generated, Electroporation

    Effects of wall-weakening agents on the electroporation efficiency of B. subtilis ZK. Various weakening agents at different concentration gradients (0.25%–0.85% glycine ( A ); 0.63%–1.56% dl -threonine ( B ); 15–110 mg/mL Tween 80 ( C ) and 1–20 μg/mL ampicillin ( D )) were separately added to the LBSP medium when the OD 600 value reached 0.5. After shaking for an additional 1 h, the electro-competent cells were prepared. One hundred nanograms of pHT43 were used for each electroporation experiment. The field strength was 20 kV·cm −1 and the electroporation buffer was TSM. The values shown are the averages from three independent experiments and the error bars indicate the standard deviations from the average values.

    Journal: International Journal of Molecular Sciences

    Article Title: Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

    doi: 10.3390/ijms16047334

    Figure Lengend Snippet: Effects of wall-weakening agents on the electroporation efficiency of B. subtilis ZK. Various weakening agents at different concentration gradients (0.25%–0.85% glycine ( A ); 0.63%–1.56% dl -threonine ( B ); 15–110 mg/mL Tween 80 ( C ) and 1–20 μg/mL ampicillin ( D )) were separately added to the LBSP medium when the OD 600 value reached 0.5. After shaking for an additional 1 h, the electro-competent cells were prepared. One hundred nanograms of pHT43 were used for each electroporation experiment. The field strength was 20 kV·cm −1 and the electroporation buffer was TSM. The values shown are the averages from three independent experiments and the error bars indicate the standard deviations from the average values.

    Article Snippet: After incubation on ice for 3 min, the mixture was loaded into a prechilled electroporation cuvette (0.1-cm electrode gap) and exposed to a single pulse generated by a Gene Pulser System (Bio-Rad, Hercules, CA, USA).

    Techniques: Electroporation, Concentration Assay

    Effects of the field strength on the electroporation efficiency of B. subtilis ZK. B. subtilis ZK was grown in LBSP medium and the electro-competent cells were prepared when the OD 600 value reached 0.85. The electroporation experiments were performed under a gradient of the field strength (8–26 kV·cm −1 ). One hundred nanograms of pHT43 plasmid were used for each electroporation experiment, and the electroporation buffer was TSM. The data shown are the averages of three independent experiments, and the error bars indicate the standard deviations from the average values.

    Journal: International Journal of Molecular Sciences

    Article Title: Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

    doi: 10.3390/ijms16047334

    Figure Lengend Snippet: Effects of the field strength on the electroporation efficiency of B. subtilis ZK. B. subtilis ZK was grown in LBSP medium and the electro-competent cells were prepared when the OD 600 value reached 0.85. The electroporation experiments were performed under a gradient of the field strength (8–26 kV·cm −1 ). One hundred nanograms of pHT43 plasmid were used for each electroporation experiment, and the electroporation buffer was TSM. The data shown are the averages of three independent experiments, and the error bars indicate the standard deviations from the average values.

    Article Snippet: After incubation on ice for 3 min, the mixture was loaded into a prechilled electroporation cuvette (0.1-cm electrode gap) and exposed to a single pulse generated by a Gene Pulser System (Bio-Rad, Hercules, CA, USA).

    Techniques: Electroporation, Plasmid Preparation

    Plots of the electroporation efficiency as a function of the DNA quantity. When the OD 600 value reached 0.5, the weakening agents (0.64% Glycine, 1.02% dl -threonine, and 0.05% Tween 80) were added to the LBSP medium and the electro-competent competent cells were prepared. B. subtilis ZK cells were transformed with various quantities of the plasmid DNA (5–500 ng). The field strength was 20 kV·cm −1 and the electroporation buffer was TSMMKK. After electroporation, the cells were incubated in a water bath at 30 or 46 °C. The electroporation experiments were repeated three times, and the data shown are the means of triplicate experiments. The error bars indicate the standard deviations from the average values.

    Journal: International Journal of Molecular Sciences

    Article Title: Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

    doi: 10.3390/ijms16047334

    Figure Lengend Snippet: Plots of the electroporation efficiency as a function of the DNA quantity. When the OD 600 value reached 0.5, the weakening agents (0.64% Glycine, 1.02% dl -threonine, and 0.05% Tween 80) were added to the LBSP medium and the electro-competent competent cells were prepared. B. subtilis ZK cells were transformed with various quantities of the plasmid DNA (5–500 ng). The field strength was 20 kV·cm −1 and the electroporation buffer was TSMMKK. After electroporation, the cells were incubated in a water bath at 30 or 46 °C. The electroporation experiments were repeated three times, and the data shown are the means of triplicate experiments. The error bars indicate the standard deviations from the average values.

    Article Snippet: After incubation on ice for 3 min, the mixture was loaded into a prechilled electroporation cuvette (0.1-cm electrode gap) and exposed to a single pulse generated by a Gene Pulser System (Bio-Rad, Hercules, CA, USA).

    Techniques: Electroporation, Transformation Assay, Plasmid Preparation, Incubation

    Effects of growth phase of B. subtilis ZK on the electroporation efficiency. B. subtilis ZK was grown in LBSP medium and the electro-competent cells were prepared at different OD 600 values (0.3–1.3). One hundred nanograms of the pHT43 plasmid were used for each electroporation experiment. The field strength was 20 kV·cm −1 and the electroporation buffer was TSM. The experiments were repeated three times. The error bars indicate the standard deviations from the average values.

    Journal: International Journal of Molecular Sciences

    Article Title: Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

    doi: 10.3390/ijms16047334

    Figure Lengend Snippet: Effects of growth phase of B. subtilis ZK on the electroporation efficiency. B. subtilis ZK was grown in LBSP medium and the electro-competent cells were prepared at different OD 600 values (0.3–1.3). One hundred nanograms of the pHT43 plasmid were used for each electroporation experiment. The field strength was 20 kV·cm −1 and the electroporation buffer was TSM. The experiments were repeated three times. The error bars indicate the standard deviations from the average values.

    Article Snippet: After incubation on ice for 3 min, the mixture was loaded into a prechilled electroporation cuvette (0.1-cm electrode gap) and exposed to a single pulse generated by a Gene Pulser System (Bio-Rad, Hercules, CA, USA).

    Techniques: Electroporation, Plasmid Preparation

    Using SCIPay to insert a chimeric antigen receptor (CAR) into the T cell receptor alpha subunit (TRAC) gene. (A) Overview of SCIPay designed to insert an anti-EGFR CAR with BFP into the human TRAC gene. (B) Outline of cell engineering strategy. After electroporation, successfully transfected cells were sorted twice to enrich BFP-positive cells. (C) BFP and TCR/TRAC expression on double-sorted population (B1). (D) Surface CD69 expression during co-culture between the indicated cell population and SKOV3 or MCF7 cells. Results in (D) represent means +/- SEM of 3 independent experiments.

    Journal: bioRxiv

    Article Title: Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering

    doi: 10.1101/2020.03.25.008276

    Figure Lengend Snippet: Using SCIPay to insert a chimeric antigen receptor (CAR) into the T cell receptor alpha subunit (TRAC) gene. (A) Overview of SCIPay designed to insert an anti-EGFR CAR with BFP into the human TRAC gene. (B) Outline of cell engineering strategy. After electroporation, successfully transfected cells were sorted twice to enrich BFP-positive cells. (C) BFP and TCR/TRAC expression on double-sorted population (B1). (D) Surface CD69 expression during co-culture between the indicated cell population and SKOV3 or MCF7 cells. Results in (D) represent means +/- SEM of 3 independent experiments.

    Article Snippet: This solution was transferred into 0.2 cm generic electroporation cuvettes (Biorad Gene Pulser; Bio-Rad laboratories, Hercules, CA) and immediately electroporated using a Lonza Nucleofector I (Lonza, Basel, Switzerland) and program X-05 (X-005 on newer Nucleofector models).

    Techniques: Electroporation, Transfection, Expressing, Co-Culture Assay

    Using SCIPay to generate a cell line that reports T cell activation. (A) Overview of SCIPay designed to insert tdTomato into the human CD69 gene. (B) Outline of cell engineering strategy. After electroporation, successfully transfected cells were sorted, activated, and sorted again to enrich PE-responding cells. (C – E) The indicated cell populations were incubated with increasing anti-CD3 and assessed for surface CD69 (C) and tdTomato (D) expression using flow cytometry. (E) Microscopy images of similar cells. (F – H) P1/J69 was transduced with an anti-CD19 CAR and cultured alongside CD19-positive cells. PE response to Nalm6 (F) and Raji (G H) co-cultures. Results in (C – H) represent means +/- SEM of 3 independent experiments.

    Journal: bioRxiv

    Article Title: Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering

    doi: 10.1101/2020.03.25.008276

    Figure Lengend Snippet: Using SCIPay to generate a cell line that reports T cell activation. (A) Overview of SCIPay designed to insert tdTomato into the human CD69 gene. (B) Outline of cell engineering strategy. After electroporation, successfully transfected cells were sorted, activated, and sorted again to enrich PE-responding cells. (C – E) The indicated cell populations were incubated with increasing anti-CD3 and assessed for surface CD69 (C) and tdTomato (D) expression using flow cytometry. (E) Microscopy images of similar cells. (F – H) P1/J69 was transduced with an anti-CD19 CAR and cultured alongside CD19-positive cells. PE response to Nalm6 (F) and Raji (G H) co-cultures. Results in (C – H) represent means +/- SEM of 3 independent experiments.

    Article Snippet: This solution was transferred into 0.2 cm generic electroporation cuvettes (Biorad Gene Pulser; Bio-Rad laboratories, Hercules, CA) and immediately electroporated using a Lonza Nucleofector I (Lonza, Basel, Switzerland) and program X-05 (X-005 on newer Nucleofector models).

    Techniques: Activation Assay, Electroporation, Transfection, Incubation, Expressing, Flow Cytometry, Microscopy, Transduction, Cell Culture