B6N(Cg)-Ngly1^{tm2d(KOMP)Wtsi}/LutzyJ

Stock number: 028975
Product name: Ngly1 KO
Research areas: Developmental Biology Research, Neurobiology Research, Research Tools

Description

This knock-out mutant of the Ngly1 gene was generated by crossing mice carrying the Ngly1^{tm2b(KOMP)Wtsi} knock-out/reporter allele to B6N.129S4-Gt(ROSA)26Sor^tm1(FLP1)Dym/J (JAX Stock#016226) mice and has exon 2 deleted. These mice may be useful for studying proteasome-mediated degradation of misfolded glycoproteins and protein processing in the endoplasmic reticulum.

Detailed description

The N-glycanase 1 (Ngly1) enzyme deglycosylates the denatured form of N-linked glycoproteins in the cytoplasm and assists their proteasome-mediated degradation. Ngly1 may play a role in the proteasome-mediated degradation of misfolded glycoproteins and protein processing in the endoplasmic reticulum. Diseases associated with NGLY1 include congenital disorder of glycosylation [type iv] and alacrimia-choreoathetosis-liver dysfunction syndrome.

These Ngly1 knock-out mice carry an allele in which exon 2 has been excised. As the mice are characterized, we will modify the strain description and add phenotype data. Homozygotes are viable and fertile. However, pups produced by homozygous females do not survive past postnatal day 1 (no milk spot is observed).

Development

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.

Allele

Symbol: Ngly1^{tm2d(KOMP)Wtsi}
Name: targeted mutation 2d, Wellcome Trust Sanger Institute
MGI accession ID: MGI:6114996
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Ngly1
Marker name: N-glycanase 1
Chromosome: 14
Strain of origin: C57BL/6N
Molecular note: The L1L2_gt2 cassette was placed upstream of the critical exon (exon 2) of the gene; inserting at bp position Chr14:16,254,154 [Build GRCm38]. The cassette is composed of composed of an FRT site, a mouse En2 intron/splice-acceptor/exon (En2 SA), a self-cleaving 2A peptide, the beta-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]; thereby flanking exon 2 with loxP sites. Cre-mediated recombination removed exon 2. Flp-mediated recombination removed the FRT-flanked neo cassette