Pvalb-T2A-dCre-D (Pvalb-2A-dCre-D) mice express a trimethoprim-inducible Cre recombinase directed to Pvalb-expressing cells by the endogenous promoter/enhancer elements of the parvalbumin locus. When induced, Cre recombinase activity is observed in scattered cells throughout the cortex, as well as restricted populations in the cerebellum, medulla, pons, pallidum, and thalamus.
Hongkui Zeng, Allen Institute for Brain Science
Genetic Background | Generation |
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Allele Type | Gene Symbol | Gene Name |
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Targeted (Recombinase-expressing, Inducible) | Pvalb | parvalbumin |
The Pvalb-T2A-dCre-D targeted mutation (also called Pvalb-2A-dCre-D, Pvalb-2A-dCre-Δ or Pvalb-2A-dCre-Δhygro) has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a destabilized Cre fusion gene (dCre) inserted in-frame at the 3' end of the parvalbumin (Pvalb) coding region. This is designed to have both endogenous gene and dCre expression directed to Pvalb-expressing cells by the endogenous promoter/enhancer elements.
The ecDHFRR12Y/Y100I domain of dCre leads to proteosomal degradation of the entire fusion protein, resulting in little or no Cre recombinase activity. Administration of the DHFR inhibitor, trimethoprim (TMP), prevents degradation of the dCre fusion gene and results in Cre recombinase activity.
When Pvalb-T2A-dCre-D mice are bred with mice containing loxP-flanked sequences, TMP-stabilized Cre recombination will result in deletion of floxed sequences in the Pvalb-expressing cells of the double mutant offspring.
Specifically, the donating investigator reports that Pvalb-T2A-dCre-D mice have trimethoprim-inducible Cre recombinase expression (in situ hybridization) that is specific and efficient in scattered cells throughout the cortex, as well as restricted populations in the cerebellum, medulla, pons, pallidum, and thalamus.
In the absence of trimethoprim, these same regions exhibit significantly reduced Cre recombinase activity (highest expression in particular cells of the cerebellum and in the thalamic reticular nucleus). Heterozygous mice are viable and fertile with no gross physical or behavioral abnormalities. The donating investigator did not examine dCre activity in tissues other than brain, and did not attempt to generate homozygous mice to date (July 2013).
For characterization information, see images at the Allen Institute for Brain Science website (Pvalb-T2A-dCre images).
The dCre fusion gene (also called destabilized Cre, hDHFR/Cre or ecDHFRR12Y/Y100I/Cre) is Cre recombinase fused at its N terminus to the first 159 amino acids of the Escherichia coli K-12 strain chromosomal dihydrofolate reductase gene (DHFR or folA) harboring the G67S mutation and modified to also include the R12Y/Y100I destabilizing domain mutations. The ecDHFRR12Y/Y100I domain of dCre leads to proteosomal degradation of the entire fusion protein, resulting in little or no Cre recombinase activity. The donating investigator reports that administration of the high affinity DHFR inhibitor trimethoprim (TMP; at a concentration of 0.25-0.30 mg/g body weight) prevents degradation of the dCre fusion gene, resulting in Cre recombinase activity.
The Pvalb-T2A-dCre-D targeted mutation (also called Pvalb-2A-dCre-D, Pvalb-2A-dCre-Δ or Pvalb-2A-dCre-Δhygro) was created in the laboratory of Dr. Hongkui Zeng (Allen Institute for Brain Science) to have a T2A sequence and a destabilized Cre fusion gene (dCre) 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-hDHFR/Cre" vector and a FLP-expressing plasmid to facilitate recombination.
Correctly re-targeted ES cells had (from 5' to 3')
a partial Pvalb intron 3 sequence containing an frt3 site,
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 dCre fusion gene (described below) that is in-frame with the Pvalb coding sequence,
the Pvalb 3' UTR sequence from exon 4,
an AttB site,
a PGK-hygromycin-SV40polyA cassette (with an mRNA splice donor-frt5 site-mRNA splice acceptor in the hygromycin gene),
and an AttP site.
The dCre fusion gene (also called destabilized Cre, hDHFR/Cre or ecDHFRR12Y/Y100I/Cre) is Cre recombinase fused at its N terminus to the first 159 amino acids of the Escherichia coli K-12 strain chromosomal dihydrofolate reductase gene (DHFR or folA) harboring the G67S mutation and modified to also include the R12Y/Y100I destabilizing domain mutations.
Correctly re-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 sequences (PGK-5'hygro::mRNA splice donor::frt5::mRNA splice acceptor::3'hygro::SV40 polyA) and replace it with the recombined AttB/AttP site (AttL). The resulting Pvalb-T2A-dCre-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed) prior to sending generation N6F1 males to The Jackson Laboratory Repository in 2013. Upon arrival, males were used to cryopreserve sperm. To establish the living Pvalb-T2A-dCre-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Expressed Gene | cre/folA, cre/folA, |
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Site of Expression | With trimethoprim induction, cre expression (by (in situ hybridization detection) is efficient in scattered cells throughout the cortex, as well as restricted populations in the cerebellum, medulla, pons, pallidum, and thalamus. |
Allele Name | targeted mutation 5.1, Hongkui Zeng |
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Allele Type | Targeted (Recombinase-expressing, Inducible) |
Allele Synonym(s) | Pvalb-2A-dCre; Pvalb-2A-dCre-DeltaHygrO; Pvalb-2A-hDHFR/Cre-D; Pvalb-2A-hDHFR/Cre-DeltaHygro |
Gene Symbol and Name | Pvalb, parvalbumin |
Gene Synonym(s) | |
Promoter | Pvalb, parvalbumin, mouse, laboratory |
Expressed Gene | cre/folA, cre/folA, |
Site of Expression | With trimethoprim induction, cre expression (by (in situ hybridization detection) is efficient in scattered cells throughout the cortex, as well as restricted populations in the cerebellum, medulla, pons, pallidum, and thalamus. |
Strain of Origin | (129S6/SvEvTac x C57BL/6NCrl)F1 |
Chromosome | 15 |
Molecular Note | ES cells previously targeted with a T2A-Cre vector immediately downstream of the parvalbumin translational STOP codon were re-targeted with a "T2A-hDHFR/Cre" vector and a FLP-expressing plasmid to facilitate recombination. Correctly re-targeted ES cells had (from 5' to 3') a partial Pvalb intron 3 sequence containing an frt3 site, a partial Pvalb exon 4 sequence up to (but not including) the endogenous stop codon, a T2A in-frame with the Pvalb coding sequence, a dCre fusion gene that is in-frame with the Pvalb codingsequence, the Pvalb 3' UTR sequence from exon 4, an AttB site, a PGK-hygromycin-SV40polyA cassette (with an mRNA spice donor-frt5 site-mRNA spice acceptor in the hygromycin gene), and an AttP site. The dCre fusion gene ( destabilized Cre, hDHFR/Cre or ecDHFR |
When maintaining a live colony, heterozygous mice may be bred to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). The phenotype of homozygous mice has not yet been determined (July 2013).
When using the Pvalb-T2A-dCre-D , Pvalb-2A-dCre-D mouse strain in a publication, please cite the originating article(s) and include JAX stock #022863 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
---|---|---|
Heterozygous or Wildtype for Pvalb<tm5.1(cre/folA)Hze> |
Frozen Mouse Embryo | B6.Cg-Pvalb<tm5.1(cre/folA)Hze>/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | B6.Cg-Pvalb<tm5.1(cre/folA)Hze>/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | B6.Cg-Pvalb<tm5.1(cre/folA)Hze>/J Frozen Embryo | $3373.50 |
Frozen Mouse Embryo | B6.Cg-Pvalb<tm5.1(cre/folA)Hze>/J Frozen Embryo | $3373.50 |
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