B6(SJL)-Mthfr^em1Adiuj/J

Stock number: 031464
Product name: Mthfr^677C>T
Research areas: Mouse/Human Gene Homologs, Neurobiology Research

Description

Mthfr^677C>T carries a mutant of the Mthfr gene containing an A262V amino acid substitution that models the human c.677C>T polymorphism. Homozygotes exhibit cerebrovascular defects. These mice may be suitable for use in studies related to Alzheimer's disease.

Detailed description

Mthfr (methylenetetrahydrofolate reductase) encodes a regulatory enzyme involved in folate and homocysteine metabolism. MTHFR generates 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine.  Mthfr^677C>T (TT) knockin mice were created using CRISPR/cas9 genome editing to express the A267V (alanine to valine, c.806C>T) amino acid substitution. c.806C>T is orthologous to human c.677C>T, a patient mutation identified in GWAS studies that is associated with increased risk for Alzheimer's disease (AD) and vascular dementia. Mice homozygous for the mutation are viable, homozygous fertility has not been assessed. Homozygous TT mice exhibit decreased MTHFR enzyme activity (as well as decreased thermostability) in liver extracts from 3-4 month old mice, increased homocysteine levels (peaking in 6 month old mice), decreased protein levels in the brain (at 3-4 months) as well as decreased metabolites within the folate and methionine pathways (females have lower levels than males). Analysis of cerebrovascular morphology in homozygotes reveals vessels with closed or thickened lumen, degenerating cells (with an apoptotic phenotype), swollen astrocyte endfeet as well as an increased number of activated microglia/monocytes. Heterozygotes have a less severe phenotype. These mice may be suitable for use in studies related to Alzheimer's disease.

Development

CRISPR/Cas9 genome editing was used to create an amino acid substitution at position 262 (alanine to valine, c.806C>T ENSMUST00000069604.15, A262V) in the Mthfr locus on Chromosome 4. Single guide RNA and Cas9 endonuclease were introduced into the cytoplasm B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J (Stock No. 028709)-derived fertilized eggs with well recognized pronuclei. Embryos were transferred to pseudopregnant females, and correctly targeted pups were bred to NOD/ShiLtJ for germline transmission. Correctly targeted pups were identified by sequencing and PCR. Mice were bred to C57BL/6J for at least 4 generations and the Apoe^{tm1.1(APOE*4)Adiuj} and Trem2^em1Adiuj alleles were bred out of the colony. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Mthfr^em1Adiuj
Name: endonuclease-mediated mutation 1, MODEL-AD Center
MGI accession ID: MGI:6202965
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Synonyms: Mthfr^677CT
Marker symbol: Mthfr
Marker name: methylenetetrahydrofolate reductase
Chromosome: 4
Strain of origin: B6(SJL)-Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj/J
Molecular note: CRISPR/Cas9 genome editing is used to introduce a point mutation (GCC to GTC) creating the A262V mutation. This C to T mutation is homologous to the human C677T polymorphism that has been associated with various diseases including Alzheimer's, vascular and metabolic disease.