B6;SJL-Slc6a17^em1Adiuj Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J

Stock number: 031948
Product name: B6J-APOE4/Trem2*R47H/Slc6a17*P61P SNP
Research areas: Cardiovascular Research, Metabolism Research, Neurobiology Research, Research Tools

Description

The B6J-APOE4/Trem2*R47H/Slc6a17*P61P SNP triple mutant strain carries a humanized ApoE knock-in mutation (sequence coding for isoform E4), a CRISPR/cas9-generated knock-in P61P point mutation of the Slc6a17 gene 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.

Detailed description

This triple mutant strain carries 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); a knock-in P61P point mutation of the Slc6a17 gene; and a knock-in of a point mutation into the mouse Trem2 (triggering receptor expressed on myeloid cells 2) gene containing a R47H point mutation, with two silent mutations. 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 Slc6a17 (solute carrier family 6 (neurotransmitter transporter), member 17 ) gene encodes a sodium-dependent neurotransmitter and neutral amino acid transporter. The targeted Trem2 gene 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 homozygous for the Apoe^{tm1.1(APOE*4)Adiuj} and Trem2^em1Adiuj alleles, and heterozygous for the Slc6a17^em1Adiuj allele are viable and fertile. Homozygous viability/fertility has not been tested for the Slc6a17^em1Adiuj allele (June 2019). As the mice are characterized, we will modify the strain description and add phenotype data.

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 triple mutant line was generated by utilizing CRISPR/cas9 endonuclease mediated genome editing of the Slc6a17 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 (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 (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 Slc6a17^em1Adiuj allele, plasmids encoding a single guide RNA designed to introduce a P61P point mutation (CCA to CCG) into the Slc6a17 gene and the cas9 nuclease were introduced into the cytoplasm of B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J (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 B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J (Stock No. 028709) for 2 generations. Sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J oocytes (Stock No. 028709).

Allele

Symbol: Trem2^em1Adiuj
Name: endonuclease-mediated mutation 1, MODEL-AD Center
MGI accession ID: MGI:5770794
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Synonyms: Jax R47H ki; Trem2 R47H KI
Marker symbol: Trem2
Marker name: triggering receptor expressed on myeloid cells 2
Marker synonyms: AD17; PLOSL2; TREM-2; Trem2a; Trem2b; Trem2c; Trem2-Mia; Trem2-Mib
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: Slc6a17^em1Adiuj
Name: endonuclease-mediated mutation 1, AD-Model Center
MGI accession ID: MGI:6316319
Generation method: Endonuclease-mediated
Attributes: Not Specified
Synonyms: Slc6a17^P61P
Marker symbol: Slc6a17
Marker name: solute carrier family 6 (neurotransmitter transporter), member 17
Marker synonyms: D130012J15Rik; MRT48; NTT4
Chromosome: 3
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 P61P point mutation (CCA to CCG) into the Slc6a17 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
Synonyms: APOE^epsilon4
Marker symbol: Apoe
Marker name: apolipoprotein E
Marker synonyms: AD2; APO-E; ApoE4; APOEA; LDLCQ5; LPG
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.