B6.Cg-Tardbp^em9Lutzy Apoe^{tm1.1(APOE*4)Adiuj} App^em5Msasn/KmsoJ

Stock number: 034543
Product name: Tardbp^G348C ; APOE4 KI ; hAbeta^Swe
Strain synonyms: Tardbp G348C KI ; APOE*4 KI ; App KI (Swe)
Research areas: Cardiovascular Research, Metabolism Research, Mouse/Human Gene Homologs, Neurobiology Research, Research Tools

Description

The Tardbp^G348C ; APOE4 KI ; hAbeta^Swe quadruple mutant strain harbors several mutations associated with human neurodegenerative disease.
Tardbp^G348C encodes a mutation found in Lou Gehrig's disease (Amyotrophic lateral sclerosis; ALS) patients.
APOE4 KI is a humanized ApoE knock-in mutation encoding isoform E4 suitable for use in studying lipoproteins, arteriosclerosis and coronary heart disease.
hAbeta^Swe harbors Swe mutations (K595N, M596L) and 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) associated with Alzheimer's disease.

Detailed description

Stock No. 034543 Tardbp^G348C ; APOE4 KI ; hAbeta^Swe (aka, Tardbp G348C KI ; APOE*4 KI ; App KI (Swe)) is a triple mutant strain harboring several mutations associated with human neurodegenerative disease. Mice homozygous for all three mutant alleles are viable and fertile [January 2025]. If additional characterization of these mice is performed, we may modify the strain description accordingly. Each allele is described below.

The Tardbp^G348C allele encodes a mutation found in Lou Gehrig's disease (Amyotrophic lateral sclerosis; ALS) patients. The endogenous Tardbp gene encodes the DNA and RNA-binding protein TDP-43. Ubiquinated accumulations of mutant TDP-43 have been linked to the development of ALS and frontotemporal lobar degeneration.

The APOE4 KI humanized knock-in allele has mouse Apoe exons 2, 3 and most of exon 4 replaced by a human APOE4 gene sequence including exons 2, 3 and 4 (and some 3' UTR sequence). This knock-in allele encodes apolipoprotein isoform E4, which is important in lipoprotein metabolism and cardiovascular disease as well as Alzheimer's disease, immunoregulation and cognition.

The hAbeta^Swe allele harbors Swe mutations (K595N, M596L) and 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) associated with Alzheimer's disease. The endogenous App gene encodes amyloid beta precursor protein, a transmembrane cell surface receptor that is cleaved by secretases.

Development

Stock No. 034543 Tardbp^G348C ; APOE4 KI ; hAbeta^Swe (aka, Tardbp G348C KI ; APOE*4 KI ; App KI (Swe)) is a triple mutant strain assembled in the laboratory of Dr. Kristen O'Connell (The Jackson Laboratory) by breeding together multiple C57BL/6J-congenic strains and selecting for the mutant alleles listed below.

The Tardbp^G348C (Tardbp G348C KI) allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing by Dr. Cathleen Lutz (The Jackson Laboratory). Plasmids encoding a signal guide RNA were designed to introduce a glycine-to-cysteine G348C mutation in the TAR DNA binding protein locus (Tardbp) on Chromosome 4. These sequences and Cas9 nuclease were introduced into the cytoplasm C57BL/6J-derived fertilized eggs with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Progeny were screened by DNA sequencing of the targeted region, which identified a founder mouse harboring the desired allele. This founder was bred to C57BL/6J mice (Stock No. 000664) for germline transmission. The Tardbp^G348C colony was thereafter maintained on the C57BL/6J genetic background (see Stock No. 028435).

The APOE4 KI (APOE*4 KI) allele was generated using homologous targeting by Dr. Mike Sasner (The Jackson Laboratory) and MODEL-AD. A targeting vector containing a 3' FRT-flanked PGK-neo cassette was used to replace the mouse apolipoprotein E (Apoe) 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->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 C57BL/6J-congenic FLP deleter mice (Stock No. 005703) to remove the FRT-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. The APOE4 KI colony was thereafter maintained on the C57BL/6J genetic background (see Stock No. 027894).

The hAbeta^Swe (App KI (Swe)) allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing by Dr. Mike Sasner (The Jackson Laboratory). Guide RNAs were selected to target exon 14 of the amyloid beta precursor protein locus (App) on Chromosome 16. Donor DNAs were created encoding lysine-to-asparagine K595N (AAG->AAT), methionine-to-leucine M596L (ATG->CTG), glycine-to-arginine G601R (GGA->CGA), phenylalanine-to-tyrosine F606Y (TTT->TAT) and arginine-to-histidine R609H (CGC->CAT) mutations. Ensembl transcript ID ENSMUST00000005406.10 (App-201) was used as reference for the exon numbering and the guide/donor sequences. These sequences and Cas9 nuclease were introduced into C57BL/6J-congenic Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj (Stock No. 028709)-derived fertilized eggs with well recognized pronuclei, and then transferred to pseudopregnant females. Progeny were screened by DNA sequencing of the targeted region, which identified a founder mouse harboring the desired allele. This founder was bred to C57BL/6J-congenic Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj Trem2^em1Adiuj mice (Stock No. 030670) to develop the colony. The hAbeta^Swe colony was thereafter maintained on the C57BL/6J genetic background. The Apoe^{tm1.1(APOE*4)Adiuj}, App^em1Adiuj and Trem2^em1Adiuj alleles were subsequently bred away.
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: Tardbp^em9Lutzy
Name: endonuclease-mediated mutation 9, Cathy Lutz
MGI accession ID: MGI:6295441
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Synonyms: Tardbp^G348C
Marker symbol: Tardbp
Marker name: TAR DNA binding protein
Marker synonyms: 1190002A23Rik; ALS10; Tdp43; TDP-43
Chromosome: 4
Strain of origin: C57BL/6J
Molecular note: CRISPR/Cas9 genome editing is used to introduce a G348C amino acid substitution. In humans, the G348C substitution is associated with amyotrophic lateral sclerosis.

Allele

Symbol: App^em5Msasn
Name: endonuclease-mediated mutation 5, Michael Sasner
MGI accession ID: MGI:8166668
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: The allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing by using guide RNAs selected to target exon 14 of the amyloid beta precursor protein locus (App) on Chromosome 16. Donor DNAs were created encoding lysine-to-asparagine K595N (AAG->AAT), methionine-to-leucine M596L (ATG->CTG), glycine-to-arginine G601R (GGA->CGA), phenylalanine-to-tyrosine F606Y (TTT->TAT) and arginine-to-histidine R609H (CGC->CAT) mutations.

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.