Casp8^em1Gne

Zygotes carrying the Casp8tm1.1Gne allele (where aspartic acid codon 387 (GAT) in exon 9 was mutated to alanine (GCC) (p.D387A) and the FRT site flanked neomycin selection gene cassette that was inserted into intron 9 was removed through subsequent flp-mediated recombination), were targeted with sgRNAs (targeting GTAAACTTTGTCTGAAGTC) and an ssODN template (CTGAGCACTTGGACATAGCCAGTGTCTGAGCTGCAGCATCAACCCCTGGATTGGGCTTGTGTTTTCCAGACCTCAGCTAAAGTTTACCAAATGAAGAACAAACCTCGGGGATACTGTCTGATCATCAACAATCATGATTTCAGCAAGGCCCGG) template using CRISPR/Cas9 technology to change aspartic acid codon 225 (GAC) in exon 8 to alanine (GCT) (p.D225A). Zygotes carrying these two alleles were then targeted with an sgRNA (targeting CAAGCTAGTGAGTCACGGGT) and an ssODN template (AGTGTGACGTTTTTTTGGTTGCTTGCAGAGATGAGCCTCAAAATGGCGGAACTGTGTGACTCGCCAAGAGAACAAGCTAGTGAGTCACGCGTAGGTGTGTCTCCTACCTCTCTCTTTGCATTGGTGTTCCTGTTTCCTTTGGTTGGTTCCTTT) to change aspartic acid codon 218 (GAC) in exon 8 to alanine (GCT) (p.D218A). (J:281456)