This knock-out mutant of the Ngly1 gene was generated by crossing mice carrying the Ngly1^{tm2a(KOMP)Wtsi} knockout-first allele (JAX Stock#27582) to B6N.Cg-Tg(Sox2-cre)1Amc/J (JAX Stock#014094) mice and then to B6N.129S4-Gt(ROSA)26Sor^tm1(FLP1)Dym/J (JAX Stock#016226) mice.




The "knockout first, promoterless" Ngly1^{tm2a(KOMP)Wtsi} allele, the L1L2_gt2 cassette was placed upstream of the critical exon (exon 2) of the N-glycanase 1 locus (Ngly1); insertion at bp position Chr14:16,254,154 [Build GRCm38]. The L1L2_gt2 cassette is composed of an FRT site, a mouse En2 intron/splice-acceptor/exon (En2 SA), a self-cleaving 2A peptide, the β-galactosidase gene (lacZ), a second self-cleaving 2A peptide, the neomycin-resistance gene (neo), an SV40 polyadenylation signal (SV40pA), a second FRT site and a loxP site. A second loxP site was inserted downstream of exon 2 at bp position Chr14:16,255,007 bp [Build GRCm38]; therefore flanking exon 2 with loxP sites.
The targeting vector PGS00036_A_C09 was introduced into C57BL/6N-derived JM8.F6 embryonic stem (ES) cells. Correctly targeted ES cell clone EPD0044_5_F07 from the International Knockout Mouse Consortium/International Mouse Phenotyping Consortium was injected into recipient blastocysts. The resulting chimeric animals were tested for germline transmission. Sperm was cryopreserved. Upon arrival at The Jackson Laboratory, the frozen sperm was used to fertilize oocytes from C57BL/6NJ inbred females (Stock No. 005304). Mice carrying the Ngly1^{tm2a(KOMP)Wtsi} allele were bred to B6N.Cg-Tg(Sox2-cre)1Amc/J (Stock No. 014094), generating the knock-out/lacZ reporter Ngly1^{tm2b(KOMP)Wtsi} allele. Mice carrying the Ngly1^{tm2b(KOMP)Wtsi} allele were crossed to B6N.129S4-Gt(ROSA)26Sor^tm1(FLP1)Dym/J (JAX Stock#016226) mice to excise the FRT flanked lacZ/neo cassette.

• 005304 C57BL/6NJ

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