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Fig. 1 | BMC Biotechnology

Fig. 1

From: Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

Fig. 1

CRISPR/Cas9 induced DSBs at the Rosa26 intronic XbaI site in mouse zygotes. a: Diagram of the mouse Rosa26 locus. The sgRosa26-1 target sequence upstream of the protospacer adjacent motif (PAM) and the XbaI site within the first intron are indicated. The locations of primers used for nested PCR are shown (1. PCR: R26F1/R26R1, 2. PCR: R26F2/R26R2). b: In vitro blastocyst assay: zygotes microinjected with Cas9 mRNA and sgRosa26-1 RNA were cultured for 4 days to blastocysts. Genomic DNA was extracted from each blastocyst and used for PCR amplification of the target region and genotyping by XbaI or T7 endonuclease I (T7EI). c: Agarose gel electrophoresis of 0.2 kb PCR products amplified with the R26F2/R26R2 primer pair from blastocysts derived from microinjected zygotes (25 ng/μl sgRosa26-1 and 50 ng/μl Cas9 mRNA) (top). PCR products were either digested with XbaI (middle) or with T7EI (bottom). XbaI resistant 0.2 kb or T7EI sensitive 0.1 kb bands (arrows) indicate the presence of modified Rosa26 alleles harboring sequence deletions. WT – wildtype control, M – size marker. d: Sequence comparison of cloned PCR products (from c) amplified from blastocysts #4 - #7 (from B). Deleted nucleotides are shown as dashes, the sgRosa26-1 PAM sequence is shown in red. e: Frequency of blastocysts showing NHEJ-based mutagenesis as indicated by the presence of XbaI resistant Rosa26 PCR products, in relation to the concentrations of Cas9 and sgRosa26-1 RNAs used for the microinjection of zygotes

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