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    New England Biolabs xmai
    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction <t>(PCR)</t> amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with <t>XmaI;</t> non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.
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    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Journal: The Crispr Journal

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

    doi: 10.1089/crispr.2017.0010

    Figure Lengend Snippet: Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Article Snippet: To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB).

    Techniques: CRISPR, Activity Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Knock-Out, Amplification, Mutagenesis, Sequencing

    Transgene expression cassettes and Southern analysis of selected transgenic lines. a RGA2 and b Ced9 expression cassettes. LB, left border; RB, right border. Determination of transgene copy number in c RGA2 and d Ced9 transgenic banana lines by Southern blot analysis. Genomic DNA from WT, RGA2 and Ced9 lines was digested with Hin dIII and Xma I, respectively. DNA molecular weight marker II (Roche) reference is indicated on the right hand side

    Journal: Nature Communications

    Article Title: Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4

    doi: 10.1038/s41467-017-01670-6

    Figure Lengend Snippet: Transgene expression cassettes and Southern analysis of selected transgenic lines. a RGA2 and b Ced9 expression cassettes. LB, left border; RB, right border. Determination of transgene copy number in c RGA2 and d Ced9 transgenic banana lines by Southern blot analysis. Genomic DNA from WT, RGA2 and Ced9 lines was digested with Hin dIII and Xma I, respectively. DNA molecular weight marker II (Roche) reference is indicated on the right hand side

    Article Snippet: Southern blot analysis For determination of transgene copy number integration by Southern analysis, total nucleic acid was extracted from banana leaf tissue, treated with RNAse A, and 15 μg aliquots of genomic DNA were digested overnight with 20 U of restriction enzyme Hin dIII or Xma I (New England Biolabs) overnight at 37 °C.

    Techniques: Expressing, Transgenic Assay, Southern Blot, Molecular Weight, Marker

    Labeling of extracellular tetracysteine-tagged proteins by membrane-impermeant biarsenical dyes. Wide-field images of HEK293 cells transiently transfected with FLNCCPGCCMEP-Myc-V2R-CeFP vasopressin receptor and stained for 10 min with the following: (top row) 2.5 µM sulfo-FlAsH (sFlAsH)-EDT 2 ) and 10 µM EDT after a 30-min preincubation with 5 mM 2-mercaptoethanesulfonate (MES); (middle row) 2.5 µM sFlAsH-EDT 2 and 10 µM EDT after a 30-min preincubation with 0.5 mM tris(carboxyethyl)phosphine (TCEP) and 5 mM MES; (bottom row) 2.5 µM ReAsH-EDT 2 and 5 µM 2,3-dimercaptopropane-sulfonate (DMPS) after a 30-min preincubation with 0.5 mM TCEP and 5 mM MES. Reducing agents were retained during staining and removed with unbound dye by washing with 100 µM DMPS for 10 min before imaging. All procedures were carried out at 37 °C in HBSS/glucose. DIC, differential interference contrast. The white scale bar = 10 µm.

    Journal: Nature protocols

    Article Title: Fluorescent labeling of tetracysteine-tagged proteins in intact cells

    doi: 10.1038/nprot.2010.129

    Figure Lengend Snippet: Labeling of extracellular tetracysteine-tagged proteins by membrane-impermeant biarsenical dyes. Wide-field images of HEK293 cells transiently transfected with FLNCCPGCCMEP-Myc-V2R-CeFP vasopressin receptor and stained for 10 min with the following: (top row) 2.5 µM sulfo-FlAsH (sFlAsH)-EDT 2 ) and 10 µM EDT after a 30-min preincubation with 5 mM 2-mercaptoethanesulfonate (MES); (middle row) 2.5 µM sFlAsH-EDT 2 and 10 µM EDT after a 30-min preincubation with 0.5 mM tris(carboxyethyl)phosphine (TCEP) and 5 mM MES; (bottom row) 2.5 µM ReAsH-EDT 2 and 5 µM 2,3-dimercaptopropane-sulfonate (DMPS) after a 30-min preincubation with 0.5 mM TCEP and 5 mM MES. Reducing agents were retained during staining and removed with unbound dye by washing with 100 µM DMPS for 10 min before imaging. All procedures were carried out at 37 °C in HBSS/glucose. DIC, differential interference contrast. The white scale bar = 10 µm.

    Article Snippet: Xma I (NEB, 5,000 U, cat. no. R0180S) Sma I (NEB, 2,000 U, cat. no. R0141L) TC-FlAsH (Invitrogen, labeling kit, cat. no. T34561) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents (our FlAsH-EDT2 stock is 1 mM in DMSO; stock solutions are stored at − 20 °C) TC-ReAsH (Invitrogen, labeling kit, cat. no. T34562) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents.

    Techniques: Labeling, Transfection, Staining, Imaging

    Specific labeling with FlAsH can only be seen after appropriate washing. Confocal images of a human A 2A -adenosine receptor construct with the tetracysteine motif CCPGCC in intracellular loop 3 and CFP at the C-terminus at position Gly-340 (A 2A ) transiently expressed in HEK293 cells. Cells were imaged 48 h after transfection. The top row shows cells after labeling with 500 nM FlAsH according to the protocol without washing (thus, without Steps 11–13). The bottom row shows the same cells 10 min after addition of 250 µM EDT according to the protocol. The left column shows images excited at 430 nm and emission from 470–550 nm (i.e., CFP fluorescence); the middle column shows the same cells excited at 514 nm and emission measured from 530–600 nm (i.e., FlAsH fluorescence). After addition of EDT, the fluorescence intensity of FlAsH-labeled cells was 15- to 20-fold over background of nontransfected neighboring cells. Note that the mere addition of EDT does not represent the full washing procedure and that better background reduction is obtained with the full procedure. The right column represents the transmission images of cells. The white scale bar = 10 µm.

    Journal: Nature protocols

    Article Title: Fluorescent labeling of tetracysteine-tagged proteins in intact cells

    doi: 10.1038/nprot.2010.129

    Figure Lengend Snippet: Specific labeling with FlAsH can only be seen after appropriate washing. Confocal images of a human A 2A -adenosine receptor construct with the tetracysteine motif CCPGCC in intracellular loop 3 and CFP at the C-terminus at position Gly-340 (A 2A ) transiently expressed in HEK293 cells. Cells were imaged 48 h after transfection. The top row shows cells after labeling with 500 nM FlAsH according to the protocol without washing (thus, without Steps 11–13). The bottom row shows the same cells 10 min after addition of 250 µM EDT according to the protocol. The left column shows images excited at 430 nm and emission from 470–550 nm (i.e., CFP fluorescence); the middle column shows the same cells excited at 514 nm and emission measured from 530–600 nm (i.e., FlAsH fluorescence). After addition of EDT, the fluorescence intensity of FlAsH-labeled cells was 15- to 20-fold over background of nontransfected neighboring cells. Note that the mere addition of EDT does not represent the full washing procedure and that better background reduction is obtained with the full procedure. The right column represents the transmission images of cells. The white scale bar = 10 µm.

    Article Snippet: Xma I (NEB, 5,000 U, cat. no. R0180S) Sma I (NEB, 2,000 U, cat. no. R0141L) TC-FlAsH (Invitrogen, labeling kit, cat. no. T34561) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents (our FlAsH-EDT2 stock is 1 mM in DMSO; stock solutions are stored at − 20 °C) TC-ReAsH (Invitrogen, labeling kit, cat. no. T34562) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents.

    Techniques: Labeling, Construct, Transfection, Fluorescence, Transmission Assay