STOCK Gls2^{em1(cre/ERT2)Hzhu}/J

Stock number: 036247
Product name: Gls2-CreER
Research areas: Developmental Biology Research, Research Tools

Description

Gls2-CreER knock-in mice have the endogenous Gls2 promoter/enhancer sequences directing expression of tamoxifen-inducible Cre recombinase. These mice are a Cre-lox tool allowing inducible Cre recombination in Gls2-expressing cells/tissues (including hepatocytes surrounding the portal vein in zone 1), and may be useful in studying hepatocyte proliferation, organization and function.

Detailed description

The Gls2 gene encodes a phosphate-activated mitochondrial glutaminase highly expressed in hepatocytes, and which is a key enzyme in the conversion of glutamine to glutamate and is also involved in the elimination of reactive oxygen species.

The Gls2-CreER knock-in allele has a tamoxifen-inducible Cre recombinase (CreER^T2) inserted into of the 3' UTR of the Gls2 locus. This is designed to have the endogenous Gls2 promoter/enhancer elements directing cre/ERT2 expression primarily in hepatocytes, but not in bile ducts (see below), as well as to retain endogenous Gls2 expression. Specific details are below.

CreER^T2 fusion gene activity is inducible - observed following tamoxifen administration. As such, when Gls2-CreER mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of floxed sequences in Gls2-expressing cells of the double mutant offspring.

The donating investigator reports that Gls2-CreER mice have tamoxifen-inducible Cre recombinase activity that recapitulates the endogenous Gls2 expression pattern - observed mainly in hepatocytes surrounding the portal vein in zone 1. The donating investigator reports no CreERT2 activity (no reporter gene expression) is observed prior to tamoxifen exposure. This knock-in allele is designed to avoid disrupting expression of the endogenous protein; although Gls2 expression from the Gls2-CreER allele has not been evaluated to date (April, 2021).

Mice heterozygous for the Gls2-CreER allele are viable and fertile with no reported gross physical or behavioral abnormalities. It has not been attempted to make this strain homozygous to date (April, 2021).

The Cre-ERT2 fusion protein consists of Cre recombinase fused to a triple mutant form of the human estrogen receptor which does not bind its natural ligand (17β-estradiol) at physiological concentrations but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with lesser sensitivity, ICI 182780. Restricted to the cytoplasm, Cre-ERT2 can only gain access to the nuclear compartment after exposure to tamoxifen. To counteract the mixed estrogen agonist effects of tamoxifen injections, which can result in late fetal abortions in pregnant mice, progesterone may be coadministered.

Development

The Gls2-CreER knock-in allele (also called Gls2-IRES-CreERT2) was created in the laboratory of Dr. Hao Zhu (University of Texas Southwestern Medical Center) to have an IRES enhancer sequence and a CreER^T2 fusion gene inserted immediately downstream of the glutaminase 2 (liver, mitochondrial) locus (Gls2) on chromosome 10. The specific details are below.

The Gls2-CreER allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. Guide RNAs were selected to target the 3' UTR of the Gls2 locus. Donor DNA was selected encoding an IRES enhancer sequence, a CreER^T2 fusion gene (Cre-ER^T2; Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain), and a bovine growth hormone polyA sequence. These sequences and Cas9 nuclease were introduced into single cell C57BL/6J zygotes and transferred to pseudopregnant females. Progeny were screened by DNA sequencing to identify correctly targeted pups, which were then bred to C57BL/6J mice (Stock No. 000664) for germline transmission. The Gls2-CreER colony was then crossed to a tdTomato reporter on the C57BL/Ka background which carried the Thy1.1 allele derived from AKR. The line was then backcrossed to C57BL/6J for a total of at least two generations to remove the tdTomato reporter allele prior to sending males to The Jackson Laboratory Repository in 2021 (see SNP note below). Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

In 2021, 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 males sent to The Jackson Laboratory Repository. This revealed 4 of the 43 markers throughout the genome were not fixed for C57BL/6 allele-type (including two on chromosome 9, one of which was within the Thy1 locus, and one on chromosome 17 at the proximal end of the MHC region). In addition, 2 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating in some mice (note the allele-type for C57BL/6N are the same as other backgrounds for these 2 markers). Collectively, these data suggest that the mice sent to The Jackson Laboratory Repository were mostly C57BL/6, but contain some undetermined genetic background contributions.

Allele

Symbol: Gls2^{em1(cre/ERT2)Hzhu}
Name: endonuclease-mediated mutation 1, Hao Zhu
MGI accession ID: MGI:6704902
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing; Inducible
Synonyms: Gls2-CreER
Marker symbol: Gls2
Marker name: glutaminase 2 (liver, mitochondrial)
Marker synonyms: A330074B06Rik; GA; GLS; hLGA; Lga
Chromosome: 10
Strain of origin: C57BL/6J
Molecular note: The allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing to have an IRES enhancer sequence and a CreER(T2) fusion gene inserted immediately downstream of the glutaminase 2 (liver, mitochondrial) locus (Gls2) on chromosome 10. These sequences and Cas9 nuclease were introduced into single-cell C57BL/6J zygotes and transferred to pseudopregnant females. Progeny were screened by DNA sequencing to identify correctly targeted pups, which were then bred to C57BL/6J mice for germline transmission.