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vector pbs u6  (Thermo Fisher)


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    Thermo Fisher vector pbs u6
    Vector Pbs U6, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vector pbs u6/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    vector pbs u6 - by Bioz Stars, 2026-05
    86/100 stars

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    Genome editing of rhesus macaque pluripotent stem cells using the piggyBac system. ( a ) Schematic representation of the piggyBac vector used for CRISPR/Cas9 expression, pTT-PB-pCAG-eCas9-GFP-U6-gRNA-Neo . Three independent expression units are contained in the piggyBac construct. (1) U6 promoter regulating the expression of tRNA-gRNA, (2) CAG promoter-driven expression of eCas9-eGFP fusion transcript. A T2A sequence separates both coding sequences. (3) SV40 promoter driving the expression of the Kanamycin/Neomycin resistance gene. ( b ) Single-cell cloning work-flow. ( c ) Proof of concept mutation analysis of monoclonal lines by PCR amplification of the C-terminal TTN locus and Sanger sequencing. The figure shows the representative sequence analysis of two different clones, one of iRhpb#4 and the other Rh_ESC. Left figures show chromatograms of the wildtype versus the mutated lines. The mutation site is indicated by the appearance of doublet peaks and the gRNA binding site by a green rectangle. ( d , e ) Genome editing efficiency of the TTN regions encoding the N- and C-termini, respectively. Four (N-terminus) and five (C-terminus) independent experiments were performed. All transfections led to mutated clones with an efficiency ranging from 8.3 to 100%. Overall, mutation efficiency was 11.53% for the N-terminual gRNA and 85.3% for the C-terminual gRNA. ( d ) Graph representing editing efficiency (Mean ± SD). ( e ) Breakdown of the different experiments performed.

    Journal: Scientific Reports

    Article Title: A piggyBac -based platform for genome editing and clonal rhesus macaque iPSC line derivation

    doi: 10.1038/s41598-021-94419-7

    Figure Lengend Snippet: Genome editing of rhesus macaque pluripotent stem cells using the piggyBac system. ( a ) Schematic representation of the piggyBac vector used for CRISPR/Cas9 expression, pTT-PB-pCAG-eCas9-GFP-U6-gRNA-Neo . Three independent expression units are contained in the piggyBac construct. (1) U6 promoter regulating the expression of tRNA-gRNA, (2) CAG promoter-driven expression of eCas9-eGFP fusion transcript. A T2A sequence separates both coding sequences. (3) SV40 promoter driving the expression of the Kanamycin/Neomycin resistance gene. ( b ) Single-cell cloning work-flow. ( c ) Proof of concept mutation analysis of monoclonal lines by PCR amplification of the C-terminal TTN locus and Sanger sequencing. The figure shows the representative sequence analysis of two different clones, one of iRhpb#4 and the other Rh_ESC. Left figures show chromatograms of the wildtype versus the mutated lines. The mutation site is indicated by the appearance of doublet peaks and the gRNA binding site by a green rectangle. ( d , e ) Genome editing efficiency of the TTN regions encoding the N- and C-termini, respectively. Four (N-terminus) and five (C-terminus) independent experiments were performed. All transfections led to mutated clones with an efficiency ranging from 8.3 to 100%. Overall, mutation efficiency was 11.53% for the N-terminual gRNA and 85.3% for the C-terminual gRNA. ( d ) Graph representing editing efficiency (Mean ± SD). ( e ) Breakdown of the different experiments performed.

    Article Snippet: The pTT-PB-pCAG-eCas9-GFP-U6-gRNA-Neo vector was derived by modification of plasmid pCAG-eCAS9-GFP-U6-gRNA , which was a gift from Jizhong Zou (Addgene plasmid # 79145; http://n2t.net/addgene:79145 ; RRID: Addgene_79145).

    Techniques: Plasmid Preparation, CRISPR, Expressing, Construct, Sequencing, Clone Assay, Mutagenesis, Amplification, Binding Assay, Transfection