B6.Cg-Apoe^{tm1.1(APOE*4)Adiuj} Abca7^em2Adiuj App^em1Adiuj Trem2^em1Adiuj/J

Stock number: 036243
Product name: Abca7*A1527G/hAbeta/APOE4/Trem2*R47H
Research areas: Cardiovascular Research, Metabolism Research, Mouse/Human Gene Homologs, Neurobiology Research

Description

Abca7*A1527G/hAbeta/APOE4/Trem2*R47H is a quadruple mutant strain carrying a humanized ApoE knock-in mutation (sequence coding for isoform E4), a CRISPR/Cas9 generated Abca7 allele with an A1527G substitution, a CRISPR/Cas9-generated App allele with a humanized Abeta1-42 region (G601R, F606Y, R609H in the mouse gene, corresponding to amino acid positions 676, 681, 684 in the human APP locus) 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 quadruple 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 rs3752246 SNP knock-in mutation into the Abca7 gene (alanine to glycine at position 1527 in exon 33); a mutant allele of the App gene containing G601R, F606Y, and R609H point mutations, and a knock-in of a point mutation into 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 Abca7 gene encodes an ATP-binding cassette (ABC) transporter protein that moves lipids across membranes. Mutations in this gene have been associated with susceptibility to Alzheimer's disease. The targeted App gene encodes amyloid beta precursor protein, a transmembrane cell surface receptor that is cleaved by secretases. Mutations in this gene have been associated with Alzheimer's disease. 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}, Abca7^em2Adiuj, App^em1Adiuj, and Trem2^em1Adiuj alleles are viable and fertile. 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

Abca7*A1527G/hAbeta/APOE4/Trem2*R47H was made be crossing mice from B6.Cg-Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj Trem2^em1Adiuj/J (Stock No. 030670) to mice from B6.Cg-Apoe^{tm1.1(APOE*4)Adiuj} Abca7^em2Adiuj Trem2^em1Adiuj/J (Stock No. 030283).

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 Abca7^em2Adiuj allele: plasmids encoding a single guide RNA designed to introduce rs3752246 SNP knock-in mutation into the Abca7 gene (alanine to glycine at position 1527 in exon 33) 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) to develop the colony.

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 of 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 App^em1Adiuj allele (hAbeta): Plasmids encoding a single guide RNA designed to introduce the G601R, F606Y, and R609H point mutations into the App gene and the cas9 nuclease were introduced into the cytoplasm of Stock No. 028709-derived fertilized eggs with with well recognized pronuclei. G601R corresponds to a GGA to CGA nucleotide change, F606Y represents a TTT to TAT nucleotide mutation, and R609H incorporates a CGC to CAT nucleotide change. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and further bred to Stock No. 028709 to develop the colony.
Important note about App isoforms and exon numbering: The App-201 isoform (695 aa protein) is encoded by 16 exons, of which the Abeta sequence is encoded by exon 14. The App-206 isoform (770 aa protein) is encoded by 18 exons, of which the Abeta sequence is encoded by exon 16.

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: Abca7^em2Adiuj
Name: endonuclease-mediated mutation 2, MODEL-AD Center
MGI accession ID: MGI:6164020
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Marker symbol: Abca7
Marker name: ATP-binding cassette, sub-family A (ABC1), member 7
Chromosome: 10
Strain of origin: B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J
Molecular note: CRISPR/Cas9 genome editing methodology is used to introduce an alanine to glycine at position 1527 in exon 33.

Allele

Symbol: App^em1Adiuj
Name: endonuclease-mediated mutation 1, MODEL-AD Center
MGI accession ID: MGI:6164022
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Marker symbol: App
Marker name: amyloid beta (A4) precursor protein
Chromosome: 16
Strain of origin: B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J
Molecular note: CRISPR/Cas9 genome editing methodologies are used to introduce G601R, F606Y, and R609H point mutations into the App gene. G601R corresponds to a GGA to CGA nucleotide change, F606Y represents a TTT to TAT change, and R609H incorporates a CGC to CAT nucleotide change.

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.