Col1a1^{tm1(tetO-cas9)Sho}

KH2 embryonic stem cells (C57BL/6 x 129S4Sv/Jae), carrying the Gt(ROSA)26Sor allele were re-targeted to insert an FRT site-flanked PGK-Neo cassette and a promoterless hygromycin resistance cassette downstream of the Col1a1 locus. Correctly re-targeted ES cells were selected and the FRT site-flanked PGK-Neo downstream of the Col1a1 locus was replaced by electroporating both a "flip-in plasmid" containing a PGK promoter, ATG start codon, FRT site, a splice acceptor-double polyA sequence (SA-2xpA), tetO (tetracycline responsive element) with a CMV minimal promoter, and SV40 polyA signal, upstream of the hCas9 (human codon optimized Cas9 nuclease) sequence, and a FLPe recombinase expressing vector to facilitate tetO-hCAS9 recombination into the 3' UTR of the Col1a1 locus. (J:101977)