C57BL/6-Sirt1^em1Dahe/J

Stock number: 038608
Product name: Sirt1^cKI-H355A
Research areas: Cancer Research, Research Tools

Description

Sirt1^cKI-H355A conditional knock-in mice carry an H355A amino acid substitution preceded by a Sirt1 minigene ending in a STOP in the Sirt1 (sirtuin 1) gene. Upon exposure to Cre recombinase, the H355A mutation creates a catalytically dead allele. This strain may be useful for research in SIRT1-NOTCH1-KAT7 interactions, gene therapy, and T-cell acute lymphoblastic leukemia (T-ALL).

Of note, the donating investigator has also made their Pten enhancer knock-out (PE knock-out) mice available as Stock No. 038609.

Detailed description

The Sirt1 (sirtuin 1) gene encodes a histone deacetylase, which acts as a metabolic and epigenetic master regulator. SIRT1 deacetylates proteins involved in cancer (NOTCH1, MYC, AKT, PTEN, TRP53) as well as proteins involved in metabolism. Specifically, loss of SIRT1 in T-cell acute lymphoblastic leukemia (T-ALL), a NOTCH1-driven, rare, hematologic cancer, results in impaired progression of leukemia, while SIRT1 overexpression accelerates leukemia progression. NOTCH1 activates SIRT1 through a distal enhancer (Rr364670).

Sirt1^cKI-H355A conditional knock-in mice carry a CRISPR/Cas9 generated amino acid substitution H355A preceded by a loxP-flanked Sirt1 minigene in the Sirt1 gene. The H355A mutation creates a catalytically dead allele. Mice homozygous for the mutation are viable and fertile. Upon exposure to Cre recombinase, the minigene is removed and the H355A mutation is expressed from the endogenous Sirt1 locus. Expression of H355A in a bone marrow transplant model using retroviral transduction of ΔE-NOTCH1-GFP into mice carrying Sirt1^cKI-H355A and a cre/ERT2 with widespread expression results in increased survival time in secondary tamoxifen-treated recipients.

Development

CRISPR/Cas9 endonuclease-mediated genome editing is used to insert a loxP-flanked Sirt1 minigene that includes exons 5-9, the Sirt1 3' UTR and a STOP into intron 4 of the Sirt1 (sirtuin 1) gene on chromosome 10. In addition, a histidine to alanine amino acid substitution at codon 355 (H355A, CAT to GCT) is inserted into the endogenous exon 5. The mutation creates a catalytically dead allele. Single guide RNA, cas9 endonuclease and a the minigene template were electroporated into C57BL/6N-derived zygotes. Embryos were transferred to pseudopregnant females, and correctly targeted pups were bred to C57BL/6N mice for germline transmission and then bred to C57BL/6N for an additional 2 generations by the donating investigator (see SNP note below). Southern blot and DNA sequencing confirmed the mutation. The colony was maintained on the C57LB/6N background. Upon arrival, mice were bred to C57BL/6NJ (Stock No. 005304) for at least one generation to establish the colony.

In 2023, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Three of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating, including the rd8 recessive mutation associated with retinal degeneration on Chromosome 1 within the Crb1 locus. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Sirt1^em1Dahe
Name: endonuclease-mediated mutation 1, Daniel Herranz
MGI accession ID: MGI:7518811
Generation method: Endonuclease-mediated
Attributes: Conditional ready (e.g. floxed)
Synonyms: Sirt1^cKI-H355A
Marker symbol: Sirt1
Marker name: sirtuin 1
Marker synonyms: Sir2; SIR2alpha; SIR2L1
Chromosome: 10
Strain of origin: C57BL/6N
Molecular note: CRISPR/cas9 endonuclease-mediated genome editing is used to insert a loxP-flanked Sirt1 minigene that includes exons 5-9, the Sirt1 3' UTR and a STOP into intron 4 of the gene. In addition, a histidine to alanine amino acid substitution at codon 355 (H355A, CAT to GCT) is inserted into the endogenous exon 5. The mutation creates a catalytically dead allele.