Generation of a conditional allele by i-GONAD: design and a proof-of-principle test. (A) Schematic illustration of the strategy to generate a conditional allele by i-GONAD. The necropsy image was taken to show the anatomical location of the oviduct with a blue ink indicator. (B) Fosl1 gene structure and relative positions of ssODNA, gRNA, and genotyping primer. (ssODNA) Single-stranded oligo DNA. (C) Fosl1 genotyping results for the F1 generation. Top row detects 5′-loxP insertion by primer pair 1F&1R; bottom row detects 3′-loxP insertion by primer pair 2F&2R; 5′-loxP band size is 426 bp; 3′-loxP is 351 bp. Mouse that inherited both 5′- and 3′-loxP is highlighted in green. (Arrow) DNA marker lane, (N) negative control of PCR (water). (D) Validations of loxP sites by Sanger sequencing.
