B6.Cg-Calb1^{tm1.1(folA/cre)Hze}/J

Stock number: 023531
Product name: Calb1-2A-dgCre-D
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Calb1-2A-dgCre-D (or Calb1-T2A-dgCre-D) mice express a trimethoprim-inducible EGFP/Cre fusion gene directed by endogenous calbindin 1 promoter/enhancer elements. When induced, small-to-moderately increased Cre recombinase activity is directed at high levels to scattered cells of the cortex, hippocampus, cerebellum and striatum, and restricted cell populations in thalamus and hypothalamus.

Detailed description

The Calb1-2A-dgCre-D (or Calb1-T2A-dgCre-D) targeted mutation has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a destabilized EGFP/Cre fusion gene (dgCre) inserted downstream of the calbindin 1 translational STOP codon. This is designed to have both endogenous gene and dgCre expression directed to Calb1-expressing cells by the endogenous promoter/enhancer elements.

The ecDHFR^R12Y/Y100I domain of dgCre leads to proteosomal degradation of the entire EGFP/Cre fusion protein, resulting in reduced overall Cre recombinase activity. Administration of the DHFR inhibitor, trimethoprim (TMP), prevents degradation of the dgCre fusion gene and results in increased Cre recombinase activity. The EGFP sequences included in the DHFR-Cre cassette contribute to the destabilization of the entire dgCre fusion protein in the absence of TMP.

When Calb1-2A-dgCre-D mice are bred with mice containing loxP-flanked sequences, TMP-enhanced Cre recombination will result in deletion of floxed sequences in the Calb1-expressing cells of the double mutant offspring.

Specifically, the donating investigator reports that Calb1-2A-dgCre-D mice have trimethoprim-inducible increases in Cre recombinase activity (reporter gene expression) detected at high levels in scattered cells of the cortex, hippocampus, cerebellum and striatum, and restricted cell populations in thalamus and hypothalamus, in a pattern that correlates well with endogenous Calb1 expression. In the absence of trimethoprim, these same regions exhibit small-to-moderate reductions in Cre recombinase activity that is variable between brain regions. The donating investigator reports no EGFP fluorescence with or without TMP. Immunohistochemical detection of EGFP levels, if any, have not yet been evaluated in the presence or absence of TMP (December 2013).

Heterozygous mice are viable and fertile with no gross physical or behavioral abnormalities. The donating investigator did not examine dgCre activity in tissues other than brain, and did not attempt to generate homozygous mice to date (December 2013).

For characterization information, see images at the Allen Institute for Brain Science website (Calb1-T2A-dgCre images).

The dgCre fusion gene (also called destabilized EGFP/Cre, DHFR-EGFP-Cre, hDHFR/EGFP/Cre or ecDHFR^R12Y/Y100I/EGFP/Cre) is an enhanced green fluorescent protein/Cre recombinase fusion gene with an N terminal fusion of 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 ecDHFR^R12Y/Y100I domain of dgCre leads to proteosomal degradation of the entire fusion protein, resulting in reduced 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 dgCre fusion gene, resulting in increased Cre recombinase activity. The EGFP sequences included in the DHFR-Cre cassette contribute to the destabilization of the entire dgCre fusion protein in the absence of TMP. The donating investigator reports no EGFP fluorescence with or without TMP. Immunohistochemical detection of EGFP levels, if any, have not yet been evaluated in the presence or absence of TMP (December 2013).

Development

The Calb1-2A-dgCre-D targeted mutation (also called Calb1-T2A-dgCre-D, Calb1-2A-dgCre-Δ or Calb1-2A-dgCre-Δneo) has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a destabilized EGFP/Cre fusion gene (dgCre) inserted immediately downstream of the calbindin 1 translational STOP codon. The specific details are below.

The targeting vector contained, from 5' to 3', a partial Calb1 intron 6 through intron 10 sequence containing an frt3 site in intron 10, a partial Calb1 exon 11 sequence up to (but not including) the endogenous stop codon, a T2A sequence that is in-frame with the Calb1 coding sequence, a dgCre fusion gene (described below) that is in-frame with the Calb1 coding sequence, a bovine growth hormone polyA signal, an AttB site, a PGK-Neo-polyA cassette, an frt5 site, an mRNA splice acceptor, the 3' portion of the hygromycin gene with SV40 polyA signal, and an AttP site.

The dgCre fusion gene (also called destabilized EGFP/Cre, DHFR-EGFP-Cre, hDHFR/EGFP/Cre or ecDHFR^R12Y/Y100I/EGFP/Cre) is an enhanced green fluorescent protein/Cre recombinase fusion gene with an N terminal fusion of 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 targeting vector was electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. 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 sequences (PGK-neo-polyA::frt5::mRNA splice acceptor::3'hygro::SV40 polyA) and replace it with the recombined AttB/AttP site (AttL). The resulting Calb1-2A-dgCre-D mice were bred with C57BL/6J wildtype mice for additional generations (and the PhiC31 transgene was removed) prior to sending generation N4F1 males to The Jackson Laboratory Repository in 2013. Upon arrival, males were used to cryopreserve sperm. To establish the living Calb1-2A-dgCre-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Allele

Symbol: Calb1^{tm1.1(folA/cre)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5522769
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Calb1
Marker name: calbindin 1
Chromosome: 4
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GFP,Green Fluorescent Protein,; cre/folA,cre/folA,
Site of expression: Pattern correlates well with endogenous Calb1 expression: after trimethoprim induced Cre recombinase activity, Cre-inducible reporter allele expression is detected in scattered cells of the cortex, hippocampus, cerebellum and striatum, and restricted cell populations in thalamus and hypothalamus.
Molecular note: The targeting vector inserted a viral 2A oligopeptide (T2A, mediating ribosomal skipping) and a destabilized EGFP/Cre fusion gene (dgCre) inserted immediately downstream of the calbindin 1 translational STOP codon. The dgCre fusion gene (also called destabilized EGFP/Cre, DHFR-EGFP-Cre, hDHFR/EGFP/Cre or ecDHFR/EGFP/Cre) is an enhanced green fluorescent protein/Cre recombinase fusion gene with an N terminal fusion of 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. A PGK-neo-polyA cassette flanked by AttB and AttP sites was also part of the construct. 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 sequences and replace it with the recombined AttB/AttP site (AttL). Trimethoprim-treatment is required to stabilize the cre protein. No EGFP is detected with or without induction.