B6.129-Lrp1^tm3.1Her/J

Stock number: 041532
Product name: Lrp1^Y63F
Research areas: Cardiovascular Research, Metabolism Research, Mouse/Human Gene Homologs

Description

This LRP^Y63F knock-in (KI) strain has an engineered point-mutation allele in Lrp1 with a Y63F mutation in the distal NPxY motif [TAT>TTT in exon 89]. It may be useful for studying the physiological role of LRP in the uptake of cholesterol-rich lipoproteins from circulation and in the maintenance of plasma lipid homeostasis.

Detailed description

These LRP^Y63F mutant mice possess an engineered point-mutation, Y63F mutation in the distal NPxY motif [TAT>TTT in exon 89], in exon 1 of the targeted low-density lipoprotein (LDL) receptor-related protein 1 gene (Lrp1). The mutation selectively disables phosphorylation-dependent signaling while preserving phosphotyrosine-independent adaptor interactions, enabling uniquely clean in vivo tests of LRP1 signal transduction. Mice that are homozygous for this allele are viable, fertile, normal in size, and do not display any gross physical or behavioral abnormalities. This strain may be useful for studying the physiological role of LRP in the uptake of cholesterol-rich lipoproteins from circulation and in the maintenance of plasma lipid homeostasis.

Development

A targeting vector was designed to insert the loxP-flanked unmutated exon 88-exon 89 fragment, followed by the mutated exon 88-exon 89 fragment-neomycin cassette, which was inserted downstream of exon 87 of the murine low-density lipoprotein (LDL) receptor-related protein 1 gene, Lrp1. The TAT sequence in the distal NPxY motif of the second exon 89 encoding for tyrosine 63 was mutated to TTT coding for phenylalanine by site-directed mutagenesis (p.Y63F, c.4507A>T; 63rd amino acid of the cytoplasmic domain counted from transmembrane segment, 4507 from initiating Methionine using human transcript reference: X13916.1 for numbering the exons). For the targeting vector, the long and the short arm sequences were generated by PCR from 129 ES cell-derived DNA. The construct was electroporated into 129S7/SvEvBrd-derived JH1 embryonic stem (ES) cells. Positive clones were identified by Southern blot analysis and were injected into blastocysts to generate chimaeras. The chimeric male mice were bred to C57BL/6 female mice to establish a colony. Mutant offspring were generated by crossing with Meox-Cre transgenic mice (Stock No. 003755) to remove the first exons of 88 and 89. Mice with the LRPY63F mutation were crossed to homozygosity, and Meox-Cre was bred out, then backcrossed to C57BL/6J mice for six generations in the donor laboratory. Upon arrival at The Jackson Laboratory, sperm were cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Lrp1^tm3.1Her
Name: targeted mutation 3.1, Joachim Herz
MGI accession ID: MGI:8286585
Generation method: Targeted
Attributes: Not Applicable
Synonyms: Lrp1^Y63F
Marker symbol: Lrp1
Marker name: low density lipoprotein receptor-related protein 1
Marker synonyms: A2mr; APOER; APR; b2b1554Clo; CD91; DDH3; IGFBP3R; IGFBP-3R; IGFBP3R1; KPA; LRP; LRP-1; LRP1A; TGFBR5
Chromosome: 10
Strain of origin: 129S7/SvEvBrd
Molecular note: A loxP-flanked exon 88-exon 89 fragment followed by exon 88-neomycin-exon 89 cassette was inserted downstream of exon 87 of the murine LRP1 gene. The TAT sequence in the distal NPxY motif of the second exon 89 encoding for tyrosine 63 was mutated to TTT coding for phenylalanine by site-directed mutagenesis. Cre-recombinase removed the first exons of 88 and 89, leaving the Y63F in exon 29.