B6.Cg-Mthfr^em1Adiuj Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J

Stock number: 030922
Product name: Mthfr^677C>T/APOE4/Trem2*R47H
Research areas: Cardiovascular Research, Metabolism Research, Mouse/Human Gene Homologs, Neurobiology Research, Research Tools

Description

Mthfr^677C>T/APOE4/Trem2*R47H is a triple mutant strain carrying a CRISPR/Cas9-generated A262V point mutation of the methylenetetrahydrofolate reductase gene that models the human C677T polymorphism linked to risk of Alzheimer's disease, a humanized ApoE knock-in mutation (sequence coding for isoform E4) and a CRISPR/Cas9-generated R47H point mutation of the Trem2 gene. These mice may be suitable for use in studies related to Alzheimer's disease, lipoproteins, arteriosclerosis, and coronary heart disease.

Detailed description

This Mthfr^677C>T/APOE4/Trem2*R47H triple mutant strain carries an A262V point mutation of the Mthfr gene that models the human c.677C>T polymorphism, a humanized ApoE knock-in allele, in which exons 2, 3 and most of exon 4 of the mouse Apoe gene were replaced by human APOE4 gene sequence including exons 2, 3 and 4 (and some 3' UTR sequence) and a knock-in of a point mutation into mouse Trem2 gene containing an R47H point mutation with two silent mutations.

The Mthfr A262V point mutation models the human MTHFR c.677C>T polymorphism which is associated with an increased risk for Alzheimer's Disease. The targeted Apoe gene encodes apolipoprotein E, which is important in lipoprotein metabolism and cardiovascular disease as well as Alzheimer's disease, immunoregulation and cognition. The targeted Trem2 gene (triggering receptor expressed on myeloid cells 2) encodes a protein that is part of a receptor signaling complex with TYRO protein tyrosine kinase binding protein, and that activates macrophages and dendritic cells during immune responses. The TREM2 R47H mutation is a missense mutation in exon 2 that is one of the strongest genetic risk factors for late-onset Alzheimer's disease.

Mice that are heterozygous for Mthfr^em1Adiuj (Mthfr^677C>T), homozygous for Apoe^{tm1.1(APOE*4)Adiuj} (APOE4) and homozygous for Trem2^em1Adiuj (Trem2*R47H) are viable and fertile [March 2020]. If additional characterization of these Mthfr^677C>T/APOE4/Trem2*R47H mice is performed, we may modify the strain description accordingly.

Of note, in brains of mice homozygous for the Trem2^em1Adiuj allele (and not carrying any other mutant alleles), expression of both transcripts of Trem2 is decreased by about 50%. Mice expressing the Trem2 R47H mutation also express a novel splice variant with a deletion of 119bp at the 5' end of exon 2, due to a cryptic splice acceptor site in exon 2 (see Stock No. 027918).

Development

This Mthfr^677C>T/APOE4/Trem2*R47H triple mutant line was generated by utilizing CRISPR/Cas9 endonuclease mediated genome editing of the Mthfr gene in B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J (available as Stock No. 028709) mice.

The double mutant B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J (Stock No. 028709) was generated by crossing 2 single mutant lines.

For the Apoe^{tm1.1(APOE*4)Adiuj} allele (APOE4; available as Stock No. 027894): A targeting vector containing a 3' FRT flanked neo cassette was used to replace exons 2, 3 and most of exon 4 with 1.5 kb of human APOE gene sequence including exons 2, 3 and 4 (and some 3' UTR sequence). The human ApoE sequence also contains a 2 nucleotide A to T substitution in intron 3 for identification of the targeted allele by Southern analysis. The construct was electroporated into C57BL/6J derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission by crossing to C57BL/6J. Heterozygous animals were crossed subsequently to FLP recombinase expressing mice (see Stock No. 005703) to remove the FRT site flanked PGK-neo cassette. Mice that no longer contained the FRT flanked PGK-neo cassette were then backcrossed to C57BL/6J at least once to remove the FLP recombinase transgene.

For the Trem2^em1Adiuj allele (Trem2*R47H; available as Stock No. 027918): Plasmids encoding a single guide RNA designed to introduce a R47H point mutation, with two silent mutations, into the Trem2 gene and the cas9 nuclease were introduced into the cytoplasm C57BL/6J-derived fertilized eggs with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and further bred to C57BL/6J (Stock No. 000664) to develop the colony.

For the Mthfr^em1Adiuj allele (Mthfr c.677C>T): Plasmids encoding a single guide RNA are designed to introduce a point mutation (GCC to GTC) into the Mthfr gene creating the A262V mutation. Plasmids and cas9 nuclease were introduced into the cytoplasm Stock No. 028709-derived fertilized eggs with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and further bred to Stock No. 028709 for 2 generations to develop the colony. Sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize oocytes from Stock No. 028709 females.

Allele

Symbol: Trem2^em1Adiuj
Name: endonuclease-mediated mutation 1, MODEL-AD Center
MGI accession ID: MGI:5770794
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Marker symbol: Trem2
Marker name: triggering receptor expressed on myeloid cells 2
Chromosome: 17
Strain of origin: C57BL/6J
Molecular note: The allele was generated by injecting cas9 nuclease and a single guide RNA designed to introduce a R47H point mutation with two silent mutations (lysine AAG>AAA and alanine GCC>GCA) into the gene.

Allele

Symbol: Apoe^{tm1.1(APOE*4)Adiuj}
Name: targeted mutation 1.1, MODEL-AD Center
MGI accession ID: MGI:5810209
Generation method: Targeted
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Apoe
Marker name: apolipoprotein E
Chromosome: 7
Strain of origin: C57BL/6J
Expressed genes: APOE,apolipoprotein E,human
Site of expression: Endogenous mouse elements direct human APOE gene sequence (including exons 2, 3 and 4 and some 3' UTR sequence).
Molecular note: The targeting vector contains a 3' FRT flanked neo cassette and 1.5 kb of human APOE gene sequence including exons 2, 3, 4 and a portion of the 3'UTR sequence (the APOE4 isoform) which is used to replace exons 2, 3 and most of exon 4 of the endogenous sequence. The human ApoE sequence carries a 2 nucleotide A to T substitution in intron 3. Flp-mediated recombination removed the FRT-flanked neo cassette.

Allele

Symbol: Mthfr^em1Adiuj
Name: endonuclease-mediated mutation 1, MODEL-AD Center
MGI accession ID: MGI:6202965
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Marker symbol: Mthfr
Marker name: methylenetetrahydrofolate reductase
Chromosome: 4
Strain of origin: B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J
Molecular note: CRISPR/Cas9 genome editing is used to introduce a point mutation (GCC to GTC) creating the A262V mutation. This C to T mutation is homologous to the human C677T polymorphism that has been associated with various diseases including Alzheimer's, vascular and metabolic disease.