C57BL/6-Trem2^{em2(TREM2*R47H)Aduci}/J

Stock number: 037497
Product name: hTREM2-R47H_KI
Research areas: Mouse/Human Gene Homologs, Neurobiology Research

Description

hTREM2*R47H_KI mice contain human TREM sequence, with a R47H mutation, replacing the corresponding mouse sequence. These mice may be useful in pre-clinical studies of Alzheimer's disease (AD).

Detailed description

Triggering receptor expressed on myeloid cells 2 (Trem2), encodes a cell surface receptor that binds polyanionic ligands including phospholipids and bacterial lipopolysaccharide. Intracellularly, TREM2 conveys signals via DAP12 (aka TYROBP) to activate myeloid lineage cells during immune responses. The rs75932628 SNP in human TREM2 introduces a R47H amino acid substitution within the extracellular Ig-like domain. TREM2*R47H is one of the strongest genetic risk factors for late-onset Alzheimer's disease (AD). In this humanized TREM2 knock-in strain, mouse genomic DNA between the initiator translation codon in exon 1 and the translation termination codon in exon 5 was replaced by the corresponding human genomic DNA sequence. The CGC codon encoding Arginine at position 47 was changed to CAC to encode Histidine. RNA-seq analysis of hippocampus and cortex in homozygous hTREM2*R47H_KI 2-month-old mice indicates that the mutant human allele is expressed at a level similar to the wild-type Trem2 allele, with no cryptic splicing product from the mutant allele.

To create the hTREM2*R47H_KI allele, mouse genomic DNA between the initiator translation codon in exon 1 and the translation termination codon in exon 5 of the Trem2 gene with the corresponding mutant human genomic DNA sequence. The human TREM2 sequence contained a CGC to CAC mutation at position 47 resulting in an arginine to histidine mutation (R47H) orthologous to the location of human SNP rs75932628. Human SNP rs75932628 has been found to be one of the strongest genetic risk factors for late-onset Alzheimer's disease (AD). If additional characterization of these hTREM2*R47H_KI mice is performed, we may modify the strain description accordingly.

Both heterozygous and homozygous mice are viable and fertile.

Development

The hTREM2*R47H_loxP targeting vector was designed by Masashi Kitazawa's lab [University of California, Irvine] to replace mouse genomic DNA between the initiator translation codon in exon 1 and the translation termination codon in exon 5 of the triggering receptor expressed on myeloid cells 2 (Trem2) gene with the corresponding mutant human genomic DNA sequence flanked by loxP sites. A frt-flanked neomycin resistance cassette was inserted in the 3' UTR. The human TREM2 sequence contained a CGC to CAC mutation at position 47 resulting in an arginine to histidine mutation (R47H) orthologous to the location of human SNP rs75932628. Human SNP rs75932628 has been found to be one of the strongest genetic risk factors for late-onset Alzheimer's disease (AD). The construct was electroporated into C57BL/6J-derived embryonic stem (ES) cells containing a FLP expression cassette. Correctly targeted ES cells were injected into blastocysts and resulting chimeric hTREM2*R47H_loxP mice, lacking the neo cassette, were bred with C57BL/6J mice (Stock No. 000664).

The Kitazawa Lab discovered that the presence of loxP sites in the hTREM2*R47H_loxP allele was associated with approximately 75% reduction in expression. To restore normal levels of hTREM2*R47H transcripts, ssODN repair template, tracrRNA and CAS9 nuclease were introduced sequentially into the cytoplasm of hTREM2*R47H_loxP-derived zygotes with well recognized pronuclei to remove both loxP sites and the remaining frt site. This also served to restore the mouse 5' UTR and 3' UTR sequences. Surviving embryos were transferred to pseudopregnant females. Progeny were screened by DNA sequencing to identify correctly targeted pups, which were then bred to C57BL/6J mice for germ line transmission. This resulting hTREM2*R47H_KI colony was backcrossed to C57BL/6J for a total of at least two generations. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Trem2^{em2(TREM2*R47H)Aduci}
Name: endonuclease-mediated mutation 2, Frank LaFerla
MGI accession ID: MGI:7327122
Generation method: Endonuclease-mediated
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Trem2
Marker name: triggering receptor expressed on myeloid cells 2
Chromosome: 17
Strain of origin: C57BL/6J-Trem2^{tm2(TREM2*R47H)Aduci}
Molecular note: The targeting vector is designed to replace mouse genomic DNA between the initiator translation codon in exon 1 and the translation termination codon in exon 5 with the corresponding mutant human genomic DNA sequence flanked by loxP sites. An FRT-flanked neomycin resistance cassette was inserted in the 3' UTR. The human TREM2 sequence contained a CGC to CAC mutation at position 47 resulting in an arginine to histidine mutation (R47H) orthologous to the location of human SNP rs75932628. Human SNP rs75932628 has been found to be one of the strongest genetic risk factors for late-onset Alzheimer's disease (AD). To ensure normal levels of TREM2*R47H transcripts, ssODN repair template, tracrRNA and CAS9 nuclease were introduced into the cytoplasm of Trem2^{tm1(TREM2*R27H)Aduci}-derived zygotes with well recognized pronuclei to remove both loxP sites and the remaining FRT site. This also served to restore the mouse 5'; UTR and 3'; UTR sequences.