Vipr2^{em1.1(cre)Hze}

Embryonic stem (ES) cells were used for CRISPR/Cas9 genome engineering. The donor vector sequences contained, from 5' to 3', a partial Vipr2 sequence spanning intron 12 up to and including the endogenous stop codon in exon 13, an internal ribosome entry site 2 sequence (IRES2; allows translation initiation in the middle of an mRNA sequence), a Cre recombinase gene, a bovine growth hormone polyA sequence, an AttB site, a PGK/gb2 promoter-Neomycin resistance gene-PGK polyA cassette, a frt5 site, an mRNA splice acceptor sequence, the 3' portion of the hygromycin gene (Hygro2) with SV40 polyA signal, and an AttP site. In addition, several basepair mutations were introduced into the first 30 nucleotides of the Vipr2 3' UTR within exon 13 to protect the donor vector from CRISPR/Cas9 cleavage. Correctly targeted ES cells (Vipr2-IRES2-Cre-neo genotype) were injected into recipient blastocysts. Chimeric mice were bred to PhiC31-expressing mice to remove the AttB/AttP-flanked sequences (PGK/gb2-Neo-pA::frt5::RNA splice acceptor::3'hygro-pA) and replace it with the recombined AttB/AttP site (AttL). (J:260362)