C57BL/6-Actb^{tm3.1(Sirt1)Npa}/J

Stock number: 013080
Research areas: Cardiovascular Research, Cell Biology Research, Developmental Biology Research, Diabetes and Obesity Research, Endocrine Deficiency Research, Internal/Organ Research, Reproductive Biology Research, Research Tools

Description

This SIRT1-KI mutant mouse strain may be useful in studies of metabolic homeostasis, physical performance and aging and lifespan.

Detailed description

Mice that are heterozygous for the targeted mutation are viable, exhibit delayed reproduction and lower body weight. No homozygotes are produced from heterozygous crosses. Fewer than expected heterozygotes are produced from heterozygote X wildtype crosses. Overexpression of gene product (protein) is detected by Western blot analysis of white adipose tissue, brown adipose tissue, MEFs, skull calvaria cells and brain tissue. Overexpression of the protein is not detected in liver or muscle tissue. Fusion gene product (mRNA) is detected in white adipose tissue by Northern blot analysis. Beta-actin protein expression is equivalent to wildtype levels. Heterozygote knock-in mice have reduced fat mass (epididymal fat pad weight), circulating free fatty acids, leptin, adiponectin and total cholesterol. Food consumption, glucose tolerance and metabolic rate (with associated higher oxygen consumption) are increased in the mutant mice. In fasted conditions, heterozygotes have lower circulating glucose levels than controls. In fed condition, heterozygotes have lower circulating insulin levels than controls.

Development

A targeting vector containing mouse SIRT1 cDNA, bovine growth hormone polyadenylation sequence, IRES sequence and floxed neo cassette was inserted into the 3' untranslated region of the mouse beta-actin locus. The construct was electroporated into C57BL/6J derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were crossed to mice expressing Cre recombinase (on the C57BL/6 background) to excise the floxed neo selection cassette. The Donating Investigator backcrossed the mice to C57BL/6 (see SNP note below) for many generations. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Actb^{tm3.1(Sirt1)Npa}
Name: targeted mutation 3.1, Novartis Pharma AG
MGI accession ID: MGI:4456082
Generation method: Targeted
Attributes: Inserted expressed sequence
Marker symbol: Actb
Marker name: actin, beta
Chromosome: 5
Strain of origin: C57BL/6J
Expressed genes: Sirt1,sirtuin 1,mouse, laboratory
Molecular note: The 3' UTR was replaced with a cassette containing an IRES sequence, floxed neo cassette, mouse Sirt1 cDNA, and a bovine growth hormone polyadenylation sequence. Cre mediated recombination removed the neo cassette. Western blot analysis confirmed expression of the cDNA in white adipose tissue, brown adipose tissue, brain, calvaria cells of the skull, and mouse embryonic fibroblasts but not in the liver or muscle. Normal expression of Actb was confirmed by western blot analysis.