B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Isr(HSA17)2Mdk App^em1Adiuj/J

Stock number: 037778
Product name: LOAD3
Strain synonyms: LOAD3, B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Tc(HSA17)2Mdk App^em1Adiuj/J
Research areas: Cardiovascular Research, Metabolism Research, Mouse/Human Gene Homologs, Neurobiology Research

Description

This strain was created by mating B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj/J (Stock No. 034415) to B6J.B6N-Tc(HSA17)2Mdk/J (Stock No. 035398).

LOAD3 mice carry a humanized ApoE knock-in mutation (sequence coding for isoform E4), the human MAPT (H1 haplotype), MAPT-AS1 transcripts and the typical MAPT protein isoforms, 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 as a base model to test late-onset risk variants and environmental risk (such as Western diet), as it expresses humanized wild-type alleles thought to be necessary but not sufficient for the development of late-onset Alzheimer's disease.

Detailed description

This LOAD3 (Late Onset Alzheimer's Disease 3) strain was created by mating B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj/J (Stock No. 034415) to B6J.B6N-Tc(HSA17)2Mdk/J (Stock No. 035398). The LOAD3 model may be used as a base model to test late-onset risk variants and environmental risk (such as Western diet), as it expresses humanized wildtype alleles thought to be necessary but not sufficient for the development of late-onset Alzheimer's disease.

The following is a description of the three alleles:

The humanized ApoE knock-in allele has exons 2, 3 and most of exon 4 of the mouse Apoe gene replaced by human APOE4 gene sequence including exons 2, 3 and 4 (and some 3' UTR sequence). Apolipoprotein E  is important in lipoprotein metabolism and cardiovascular disease as well as Alzheimer's disease, immunoregulation and cognition.

The transchromosomal (Tc) MAPT(H1)-GR (Tc(HSA17)2Mdk) mice contain a syntenic 190 kb region from human chromosome 17 (HSA17) that includes SPPLC2 (signal peptide peptidase 2C) and a modified MAPT (microtubule-associated protein tau), specifically, the H1 MAPT haplotype. The SPPL2C sequence is included because it is embedded entirely in the MAPT-AS1 transcribed region. H1 is linked with late onset Alzheimer's disease (AD) risk, progressive supranuclear palsy, Parkinson's disease, and corticobasal degeneration.

This hAbeta (App^em1Adiuj) mutant strain carries a mutant allele of the App gene containing G601R, F606Y, and R609H point mutations. 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 all three mutations are viable and fertile.

Development

This LOAD3 strain was created by mating B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj/J (Stock No. 034415) to B6J.B6N-Tc(HSA17)2Mdk/J (Stock No. 035398).

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 (hAbeta): Plasmids encoding a single guide RNA designed to introduce the G601R, F606Y, and R609H point mutations into the App gene and cas9 nuclease were introduced into the cytoplasm of B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J-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 theTrem2 allele was bred out of the colony.

The Tc(HSA17)2Mdk allele is created as follows: the mouse Sppl2c (signal peptide peptidase 2C) and Mapt (microtubule-associated protein tau) genes are replaced by human SPPL2C and MAPT (H1 haplotype) genes. The 157 kb deletion on mouse chromosome 11 stretches from, but does not include, Crhr1 to Kansl1, and is replaced by a syntenic 190 kb region from human chromosome 17 (HSA17). The deletion/insertion was made using C57BL/6N-PRX-B6N embryonic stem cells (ES); a loxN-flanked neomycin resistance cassette was removed by transient cre expression prior to establishing the mouse line. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were crossed to C57BL/6J females and maintained on the C57BL/6J background.

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
Marker synonyms: AAA; Abeta; ABPP; AD1; Adap; alpha-sAPP; APPI; appican; betaAPP; CTFgamma; Cvap; E030013M08Rik; PN2; PN-II; preA4; protease nexin II
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
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.

Allele

Symbol: Isr(HSA17)2Mdk
Name: interspecies replacement, human Chr 17, Michael Koob 2
MGI accession ID: MGI:6705045
Generation method: Targeted
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: MAPT(H1.0)-GR
Marker symbol: Isr(HSA17)2Mdk
Marker name: interspecies replacement, human Chr 17, Michael Koob 2
Marker synonyms: Tc(HSA17)2Mdk
Chromosome: 11
Strain of origin: C57BL/6NTac
Molecular note: A 157 kb deletion on mouse chromosome 11 stretching from, but not including, Crhr1 to Kansl1 is replaced by a syntenic 190 kb region from human chromosome 17 (HSA17). The mouse Sppl2c (signal peptide peptidase 2C) and Mapt (microtubule-associated protein tau) genes are replaced by human SPPL2C and MAPT (H1 haplotype) genes. A loxN-flanked neomycin resistance cassette was removed by transient cre expression.