STOCK Apoc4^{em1(cre/ERT2)Hzhu}/J

Stock number: 036343
Product name: Apoc4-CreER
Research areas: Developmental Biology Research, Research Tools

Description

Apoc4-CreER knock-in mice have the endogenous apolipoprotein C-IV promoter/enhancer sequences directing expression of tamoxifen-inducible Cre recombinase. These mice are a Cre-lox tool allowing inducible Cre recombination in Apoc4-expressing cells/tissues (including hepatocytes of all zones surrounding the central vein), and may be useful in studying hepatocyte proliferation, organization and function.

Detailed description

The Apoc4 gene encodes the apolipoprotein C-IV protein which is expressed in hepatocytes of all zones.

The Apoc4-CreER knock-in allele has a tamoxifen-inducible Cre recombinase (CreER^T2) inserted into of the 3' UTR of the Apoc4 locus. This is designed to have the endogenous Apoc4 promoter/enhancer elements directing cre/ERT2 expression primarily in hepatocytes, but not in bile ducts (see below), as well as to retain endogenous Apoc4 expression. Specific details are below.

CreER^T2 fusion gene activity is inducible - observed following tamoxifen administration. As such, when Apoc4-CreER mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of floxed sequences in Apoc4-expressing cells of the double mutant offspring.

The donating investigator reports that Apoc4-CreER mice have tamoxifen-inducible Cre recombinase activity that recapitulates the endogenous Apoc4 expression pattern - observed in hepatocytes of all zones. 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 Apoc4 expression from the Apoc4-CreER allele has not been evaluated to date (April, 2021).

Mice heterozygous for the Apoc4-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 Apoc4-CreER knock-in allele (also called Apoc4-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 pyruvate kinase liver and red blood cell locus (Apoc4) on chromosome 7. The specific details are below.

The Apoc4-CreER allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. Guide RNAs were selected to target the 3' UTR of the Apoc4 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 Cyp1a2-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 3 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). In addition, 2 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating (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: Apoc4^{em1(cre/ERT2)Hzhu}
Name: endonuclease-mediated mutation 1, Hao Zhu
MGI accession ID: MGI:6704909
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing; Inducible
Synonyms: Apoc4-CreER
Marker symbol: Apoc4
Marker name: apolipoprotein C-IV
Marker synonyms: Acl; APO-CIV; APOC-IV
Chromosome: 7
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 pyruvate kinase liver and red blood cell locus (Apoc4) on chromosome 7. 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.