The pRosa-Cas9 targeting vector, the template for homology-directed repair (HDR), was designed with (from 5' to 3') a 5' homology arm, splice acceptor, polyA elements, a ubiquitously expressed CAG promoter, two copies of the bovine growth hormone poly(A) signal, loxP-flanked Neo/Stop cassette, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) linked to an IRES-GFP (internal ribosome entry site and Green Fluorescent Protein) reporter, and a 3' homology arm. This template construct, sgRosa26-1 guide RNA, cas9 mRNA (Cas9-162A, containing plasmid coded polyadenine (polyA) tail) were microinjected into C57BL/6NCrl (Charles River, Sulzbach, Germany) zygotes, which had just been supplemented with Cas9 protein. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and bred to C57BL/6J (Stock No. 000664) once, generating the floxed stop ROSALSL-Cas9 line (see Stock No. 028551). The ROSALSL-Cas9 mice were then crossed to a human cytomegalovirus minimal promoter driven Cre recombinase strain (Stock No. 006054) to excise the floxed STOP cassette. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, all 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.
