Fig. 5
From: i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases
Generation of reporter knock-in mice using the i-GONAD method. a Schematic diagram showing insertion of T2A-mCitrine cassette into Pitx3 locus. The target sequence and the genotyping primer sets are shown. A 925-base-long ssDNA synthesized by ivTRT method was used as the donor DNA. b mCitrine fluorescence in fetus collected at E12.5. The eye of the fetus is enlarged as an inset. c Example of genotyping analysis of knock-in G0 fetuses. Expected fragment sizes: M1035/M390 = 948 bp, M389/M1036 = 956 bp, M389/PP226 = 809 bp. N negative control, M size marker. d Representative sequencing chromatogram showing 5′ and 3′ junctional regions of the inserted cassette. The junctional sequences showing insertion derived from G0-#1 in c are shown. Red arrows indicate junctions between the arms and the genomic sequences. e Genome editing efficiency of the Pitx3 locus by the i-GONAD method