B6.Cg-Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj Cd2ap^em1Adiuj Trem2^em1Adiuj/J

Stock number: 033873
Product name: Cd2ap promoter SNP/hAbeta/APOE4/Trem2*R47H
Research areas: Cardiovascular Research, Metabolism Research, Mouse/Human Gene Homologs, Neurobiology Research, Research Tools

Description

Cd2ap promoter SNP/hAbeta/APOE4/Trem2*R47H mice carry a CRISPR/Cas9-generated T-to-A point mutation in the promoter of the CD2-associated protein locus. This models the Alzheimer's disease non-coding risk variant rs4715019.
This mutation is maintained with the hAbeta/APOE4/Trem2*R47H mutations (Stock No. 030670) intended to increase risk of late-onset Alzheimer's disease: a humanized Apoe knock-in mutation (sequence coding for human isoform E4), 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.

Detailed description

This Cd2ap promoter SNP/hAbeta/APOE4/Trem2*R47H 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 mutant allele of the App gene containing G601R, F606Y and R609H point mutations, a mutant allele of the Cd2ap gene with a T-to-A point mutation in the promoter region (modeling human SNP rs4715019) and a knock-in of a point mutation into mouse Trem2 gene containing an 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 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 Cd2ap gene encodes a scaffolding protein involved in the actin cytoskeleton regulation, endocytosis and cytokinesis. Mutations of the CD2AP gene are associated with Glomerulosclerosis, focal segmental, 3. 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 homozygous for Apoe^{tm1.1(APOE*4)Adiuj} (APOE4), App^em1Adiuj (hAbeta), Cd2ap^em1Adiuj (Cd2ap promoter SNP) and Trem2^em1Adiuj (Trem2*R47H) are viable and fertile [March 2020]. If additional characterization of these Cd2ap promoter SNP/hAbeta/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 Cd2ap promoter SNP/hAbeta/APOE4/Trem2*R47H quadruple mutant line was generated by utilizing CRISPR/Cas9 endonuclease-mediated genome editing of the Cd2ap gene in C57BL/6J-congenic Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj Trem2^em1Adiuj mice (available as Stock No. 030670).
Stock No. 030670 was generated by utilizing CRISPR/Cas9 endonuclease-mediated genome editing of the App gene in C57BL/6J-congenic Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj mice (available as Stock No. 028709).

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 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 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.

For the Cd2ap^em1Adiuj allele (Cd2ap promoter SNP): plasmids encoding a single guide RNA designed to introduce a T-to-A point mutation in the promoter of the Cd2ap gene and the cas9 nuclease were introduced into the cytoplasm of Stock No. 030670-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. 030670 to develop the colony.

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: Cd2ap^em1Adiuj
Name: endonuclease-mediated mutation 1, MODEL-AD Center
MGI accession ID: MGI:6446952
Generation method: Endonuclease-mediated
Attributes: Not Specified
Marker symbol: Cd2ap
Marker name: CD2-associated protein
Chromosome: 17
Strain of origin: B6.Cg-Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj Trem2^em1Adiuj/J
Molecular note: Plasmids encoding a single guide RNA are designed to introduce a T-to-A point mutation in the promoter of the Cd2ap gene.

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.