Figure 4

Dual allele KI in ES cells could be achieved via single electroporation. (A) Schematic representation of Rosa26 locus KI strategy using two independent targeting vectors. (B) Genomic PCR analyses of ES cell clones. Red arrows in the upper or bottom panels indicate Bsd-GOI-A KI or NeoR-GOI-B, respectively. Black arrows indicate genomic DNA (gDNA) PCR control. Both 5′ and 3′ PCR positive clones are indicated with red numbers. NC1: negative control using genomic DNA from wild-type B6 mouse tail. NC2: negative control using the targeting vector. (C) Schematic representation for the combined Rosa26 and Cd6 locus KI strategy using two independent targeting vectors and gRNA-RNPs. (D,E) Genomic PCR analyses of ES cell clones. Gel images or KI ratios are shown in D or E, respectively. Red arrows indicate the 5′ or 3′ KI specific bands in Rosa26 or Cd6 loci, respectively. Both Rosa26 KI and Cd6 KI clones are indicated with red numbers. NC: negative control using genomic DNA from wild-type B6 mouse tail. (F) A schematic illustration of the genetic construct for tamoxifen-inducible Cre/loxP recombination in Rosa26 and Cd6 loci. (G) Representative double KI ES cell colonies under a fluorescent microscope without (top panels) or with (bottom panels) 4-hydroxytamoxifen (4OHT). Left panels show GFP, middle panels show RFP, and left panels show bright field. Scale bar, 50 µm. (H) A schematic illustration of tamoxifen administration followed by tissue sampling in the chimeric mice having both Rosa26 KI and Cd6 KI alleles. (I) Expression of RFP in tissues of chimeric mice. Mice were administered with tamoxifen intraperitoneally 5 times and were then used for fluorescent microscope observation.