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

Stock number: 034415
Product name: hAbeta/APOE*4
Research areas: Cardiovascular Research, Metabolism Research, Mouse/Human Gene Homologs, Neurobiology Research, Research Tools

Description

hAbeta/APOE4 mice carry a humanized ApoE knock-in mutation (sequence coding for isoform E4) and 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). These mice may be suitable for use in studies related to Alzheimer's disease, lipoproteins, arteriosclerosis, and coronary heart disease.

Detailed description

This double 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 contains G601R, F606Y, and R609H point 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.

Mice homozygous for the Apoe mutation and heterozygous for the App mutation are viable and fertile.  The viability/fertility of App mutant homozygotes has not been assessed (Oct., 2019).

Development

A triple mutant line (available as Stock No. 030670; B6.Cg-Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj Trem2^em1Adiuj/J) was generated by utilizing CRISPR/cas9 endonuclease-mediated genome editing of the App gene in B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J (available as Stock No. 028709) mice.  The Trem2 mutant allele was subsequently bred out of the triple mutant line to create this double mutant line (B6.Cg-Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj Trem2^em1Adiuj/J Stock No. 034415).

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