B6.Cg-Pvalb^{tm1.1(cre/ERT2)Hze}/J

Stock number: 021189
Product name: Pvalb-T2A-CreERT2-D , Pvalb-2A-CreERT2-D
Research areas: Neurobiology Research, Research Tools

Description

Pvalb-T2A-CreERT2-D (Pvalb-2A-CreERT2-D) mice have Cre-ERT2 fusion gene expression directed to Pvalb-expressing cells by the endogenous promoter/enhancer elements of the parvalbumin locus. These mice are a Cre-lox tool that allows inducible Cre expression of genes in Pvalb-expressing cells/tissues.

Detailed description

Mice heterozygous for the Pvalb-T2A-CreERT2-D (Pvalb-2A-CreERT2-D) allele are viable and fertile. The Pvalb-T2A-CreERT2-D targeted mutation has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a CreER^T2 fusion gene (CreERT2 fusion protein) inserted in-frame at the 3' end of the parvalbumin (Pvalb) coding region.

As such, Pvalb-T2A-CreERT2-D mice have both endogenous gene and CreERT2 fusion protein expression directed to Pvalb-expressing cells by the endogenous promoter/enhancer elements of the parvalbumin locus. CreERT2 fusion gene activity is inducible; observed following tamoxifen administration. When Pvalb-T2A-CreERT2-D mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of floxed sequences in the Pvalb-expressing cells of the double mutant offspring.

Specifically, the donating investigator reports that following tamoxifen induction, Pvalb-T2A-CreERT2-D directs reporter gene expression to scattered interneuronal populations in the cortex and hippocampus, as well as to neuronal populations in other brain regions, in a pattern that resembles endogenous Pvalb expression. Additional reporter gene expression is seen in cortical layer 5 neurons, which are unlikely to be interneurons. The donating investigator reports some CreERT2 activity prior to tamoxifen exposure (scattered CreERT2 activity in the cerebellum and thalamic reticular nucleus; fewer active cells were seen in cortex, hippocampus and striatum). The donating investigator did not examine CreERT2 activity in tissues other than brain, and did not attempt to generate homozygous mice to date (January 2013).

For characterization information, see images at the Allen Institute for Brain Science website (Pvalb-T2A-CreERT2 images).

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

Pvalb-T2A-CreERT2-D (Pvalb-2A-CreERT2-D) mice were created in the laboratory of Dr. Hongkui Zeng (Allen Institute for Brain Science) to have a T2A sequence and a CreER^T2 fusion gene inserted in-frame at the 3' end of the parvalbumin (Pvalb) coding region on chromosome 15. The specific details are below.

(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells, already targeted with a T2A-Cre vector inserted in-frame at the 3' end of the Pvalb coding region (see Stock No. 012358), were re-targeted with a "T2A-CreERT2" vector and a FLP-expressing plasmid to facilitate recombination. Correctly targeted ES cells had (from 5' to 3') a three amino acid linker sequence, an frt3 site within Pvalb intron 3, a partial Pvalb exon 4 sequence up to (but not including) the endogenous stop codon, a viral 2A oligopeptide (T2A; mediates ribosomal skipping) that is in-frame with the Pvalb coding 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), the Pvalb 3' UTR sequence from exon 4, an attB site, a PGK-hygromycin-SV40polyA cassette (with an RNA splice donor-frt5 site-RNA splice acceptor in the hygromycin gene), and an attP site.

Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to delete the attB/attP-flanked PGK-hygromycin-polyA cassette and replace it with the recombined attB/attP site (attL). The resulting Pvalb-T2A-CreERT2-D mice were bred with C57BL/6J wildtype mice for at least two additional generations (and the PhiC31 transgene was removed) prior to sending to The Jackson Laboratory Repository in 2013.

Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least three generations to create the congenic colony.

Allele

Symbol: Pvalb^{tm1.1(cre/ERT2)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5461312
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Pvalb
Marker name: parvalbumin
Chromosome: 15
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Following tamoxifen induction, Pvalb^{tm1.1(cre/ERT2)Htz} directs reporter gene expression to scattered interneuronal populations in the cortex and hippocampus, as well as to neuronal populations in other brain regions, in a pattern that resembles endogenous Pvalb expression. Additional reporter gene expression is seen in cortical layer 5 neurons, which are unlikely to be interneurons.
Molecular note: Embryonic stem (ES) cells, previously targeted with a T2A-Cre vector immediately downstream of the parvalbumin translational STOP codon were re-targeted with a T2A-CreERT2 vector and a FLP-expressing plasmid to facilitate recombination. Correctly targeted ES cells had (from 5' to 3') a three amino acid linker sequence, an frt3 site within Pvalb intron 3, a partial Pvalb exon 4 sequence up to (but not including) the endogenous stop codon, a T2A sequence that is in-frame with the Pvalb coding sequence, a CreERT2 fusion gene, the Pvalb 3' UTR sequence from exon 4, an attB site, a PGK-hygromycin-SV40polyA cassette and an attP site. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred to PhiC31-expressing mice to delete the attB/attP-flanked PGK cassette. The resulting mice were bred with C57BL/6J wildtype mice for at least two additional generations (and the PhiC31 transgene was removed).