C57BL/6J-Lyset^em1Janc/J

Stock number: 037554
Product name: Lyset KO

Research areas: Cell Biology Research

Description

Lyset knock-out mice have 184 nucleotides deletion in exon 2 of the Lyset  (lysosomal enzyme trafficking factor formerly, Tmem251) locus. Mice homozygous for the mutation exhibit increased lysosomal enzyme activity and morphological alterations in lysosomes. These mice may be useful for studies of severe lysosomal storage disorder mucolipidosis II.

Detailed description

Lyset  (lysosomal enzyme trafficking factor formerly, Tmem251) encodes a little known protein containing two transmembrane domains. Lyset colocalizes with N-acetylglucosamine-1-phosphate transferase (GNPTAB) and is involved in M6P (mannose 6-phosphate) modification, an early step in lysosomal enzyme transport. Disruption of the M6P tagging is associated with severe lysosomal storage disorder mucolipidosis II (MLII).

Lyset KO mice were created using CRISPR/cas9 genome editing to delete 184 nucleotides in exon 2 of the Lyset locus resulting in a null allele. Mice homozygous for the mutation are viable and fertile. Homozygous Lyset KO mice exhibit higher lysosomal enzyme activity (eg. α-mannosidase, β-hexosaminidase, α-L-fucosidase and β-galactosidase) than controls. Electron microscopy analysis shows accumulation of electron dense material in the cytoplasm of lysozymes as well as an increase in size in number of lysozymes, however, mice do not display obvious clinical symptoms. This strain may be useful for studies of severe lysosomal storage disorder mucolipidosis II.

Development

CRISPR/cas9 endonuclease-mediated genome editing was used to delete 184 nucleotides in exon 2 from the Lyset  (lysosomal enzyme trafficking factor formerly, Tmem251) locus on chromosome 12. The deletion results in a frameshift and loss of protein expression (Western blot analysis in brain, liver, lung, and spleen). Dual guide RNA and cas9 endonuclease were introduced into C57BL/6-derived zygotes by RNP microinjection. Embryos were transferred to pseudopregnant females, and correctly targeted pups were bred to C57BL/6J mice for germline transmission. Founders were sequenced to confirm the mutation. Mice were bred to C57BL/6J for 3 generations. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

Allele

Symbol: Lyset^em1Janc
Name: endonuclease-mediated mutation 1, Jan Carette
MGI accession ID: MGI:7384326
Generation method: Endonuclease-mediated
Attributes: Null/Knockout
Synonyms: delta184
Marker symbol: Lyset
Marker name: lysosomal enzyme trafficking factor
Marker synonyms: C14orf109; D230037D09Rik; DMAN; GCAF; RGD1566396; TMEM251
Chromosome: 12
Strain of origin: C57BL/6J
Molecular note: CRISPR/Cas9 technology targeting exon 2 generated a 184 bp deletion resulting in a frameshift. Western blot analysis confirmed absence of protein.