Generation of transgenic Cas9-based EGFP conditional KO mouse. (a) Schematic of sgRNAsEGFP-UbC-LSL-Cas9 transgenic vector. Four sgRNAs (pink) driven by U6 promoters (green arrowheads) were used to target independent sites of EGFP coding sequence. The Cas9 with nuclear localization signal (NLS) and FLAG tag was fused to LSL element and driven by UbC promoter. (b) DsRed fluorescence in organs of sgRNAs
EGFP-LSL-Cas9 mouse (Tg) line 27. The upper panel shows organs in bright field. The lower panel shows dsRed fluorescence. Dotted lines indicate the organs invisible. Scale bar, 5 mm. (c) DsRed mRNA expression level in organs of mouse line 27 was analyzed by RT-PCR. The expression level of dsRed relative to β-actin in different organs was normalized to that in brain. WT mouse RNA mixture was absent of dsRed mRNA and used as a negative control (n = 3 repeats).