The Rosa26-LSL-Cas9 targeting vector used was designed with (from 5' to 3') a 5' homology arm, a ubiquitously expressed CAG promoter, loxP-flanked 3xSV40 polyA stop cassette, a 3X-FLAG epitope tag, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) flanked by two nuclear localization signals, a P2A ribosomal skip cleavage peptide sequence, an enhanced Green Fluorescent Protein (EGFP) gene, a woodchuck hepatitis virus post-transcriptional regulatory element, bovine growth hormone polyA, pPGK-Neo-pA positive selection cassette and a 3' homology arm. The construct was electroporated into 129S-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6N blastocysts. The resulting chimeric animals were tested for germline transmission by crossing to C57BL/6N mice. The mice were then backcrossed for four generations onto C57BL/6N. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.
SNP (single nucleotide polymorphism) analysis performed as part of The Jackson Laboratory quality control effort indicates an incomplete backcross.
A 32 SNP panel analysis (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 live colony at The Jackson Laboratory Repository. Five of the 27 markers were segregating for 129. This suggests an incomplete backcross. All 5 markers that distinguish C57BL/6J from C57BL/6N were found to be segregating. This further suggests the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.
