B6.Cg-Pvalb^{tm2.1(flpe)Hze}/J

Stock number: 021191
Product name: Pvalb-2A-FlpE-D , Pvalb-T2A-FlpE-D
Research areas: Research Tools

Description

Pvalb-2A-FlpE-D (Pvalb-T2A-FlpE-D) mice have both endogenous Pvalb gene and sequence-enhanced Flp recombinase (FlpE) gene expression directed to Pvalb-expressing cells by the endogenous promoter/enhancer elements of the parvalbumin locus. These mice are a tool that allows deletion of frt-flanked sequences in Pvalb-expressing cells/tissues.

Detailed description

The Pvalb-2A-FlpE-D (Pvalb-T2A-FlpE-D) targeted mutation has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a sequence-enhanced FlpE recombinase gene inserted in-frame at the 3' end of the parvalbumin (Pvalb) coding region.

As such, Pvalb-2A-FlpE-D mice have both endogenous Pvalb gene and FlpE expression directed to Pvalb-expressing cells by the endogenous promoter/enhancer elements of the parvalbumin locus. When Pvalb-2A-FlpE-D mice are bred with mice containing frt-flanked sequences, FlpE-mediated recombination will result in deletion of frt-flanked sequences in the Pvalb-expressing cells of the double mutant offspring.

Specifically, the donating investigator reports heterozygous mice have an FlpE recombinase expression pattern in the brain that is very similar to endogenous Pvalb expression (by in situ hybridization using a FlpE-specific probe). The donating investigator did not examine FlpE activity in tissues other than brain, and did not attempt to generate homozygous mice to date (January 2013).

Mice heterozygous for the Pvalb-2A-FlpE-D allele are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities.

Development

Pvalb-2A-FlpE-D (Pvalb-T2A-FlpE-D) mice were created in the laboratory of Dr. Hongkui Zeng (Allen Institute for Brain Science) to have a T2A sequence and a sequence-enhanced FlpE recombinase 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-FlpE" 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 sequence-enhanced FlpE recombinase gene, 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-2A-FlpE-D mice were bred with C57BL/6J wildtype mice for at least seven additional generations (and the PhiC31 transgene was removed) prior to sending to The Jackson Laboratory Repository in 2013.

Upon arrival, sperm as cryopreserved. To establish the living Pvalb-2A-FlpE-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Allele

Symbol: Pvalb^{tm2.1(flpe)Hze}
Name: targeted mutation 2.1, Hongkui Zeng
MGI accession ID: MGI:5461313
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: Pvalb
Marker name: parvalbumin
Chromosome: 15
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Site of expression: Heterozygous mice have an FlpE recombinase expression pattern in the brain that is very similar to endogenous Pvalb expression.
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-FLPe (enhanced flippase) 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 sequence-enhanced flippase gene (FLPe), 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).