B6.Cg-Ccn2^{tm1.1(folA/cre)Hze}/J

Stock number: 028535
Product name: Ctgf-2A-dgCre-D
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Ctgf-2A-dgCre-D knockin mice express a trimethoprim-inducible EGFP/Cre fusion gene directed by endogenous connective tissue growth factor promoter/enhancer elements, without disrupting endogenous Ctgf expression. When induced, Cre recombinase activity is restricted to layer 6b of the cortex and in restricted populations within the cortical subplate (e.g., endopiriform nucleus). These mice may be used to generate conditional mutations for studying gain-or-loss of function and/or fate mapping in these subgroups of brain tissues.

Detailed description

The connective tissue growth factor protein encoded by the Ctgf gene is a mitogen that is secreted by vascular endothelial cells. CTGF plays a role in chondrocyte proliferation and differentiation, cell adhesion in many cell types, and is related to platelet-derived growth factor. In zebra fish, CTGF is necessary and sufficient to stimulate glial bridging and natural spinal cord regeneration.


The Ctgf-2A-dgCre-D knockin mutation has an in-frame insertion of a viral 2A oligopeptide (T2A) and a destabilized EGFP/Cre fusion gene (dgCre), all in place of the Ctgf translational STOP codon. This is designed to have both endogenous gene and dgCre expression directed to Ctgf-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 Ctgf-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 Ctgf-expressing cells of the double mutant offspring.

Specifically, the donating investigator reports that Ctgf-2A-dgCre-D mice exhibit TMP-inducible Cre recombinase activity that is specific and efficient (largely recapitulates the endogenous Ctgf expression pattern with efficient inducibility). When induced, strong Cre recombinase activity is restricted to layer 6b of the cortex and in restricted populations within the cortical subplate (e.g., endopiriform nucleus). In the absence of TMP, Cre activity is only present in a few cortical layer 6b cells. 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 2015).

Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator did not examine dgCre activity in tissues other than brain. To date (December 2015), it has not been attempted to make this strain homozygous.

For characterization information, see images at the Allen Institute for Brain Science website (Ctgf-2A-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 2015).

Development

The Ctgf-2A-dgCre-D targeted mutation (also called Ctgf-2A-dgCre-Δ or Ctgf-2A-dgCre-Δneo/hygro) has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a destabilized EGFP/Cre fusion gene (dgCre) inserted in place of the translational STOP codon of the connective tissue growth factor gene (Ctgf) on chromosome 10. The specific details are below.

The targeting vector contained, from 5' to 3', a partial Ctgf exon 1 through intron 4 sequence containing an frt3 site in intron 4, a partial Ctgf exon 5 sequence up to (but not including) the endogenous stop codon, a T2A sequence that is in-frame with the Ctgf coding sequence, a dgCre fusion gene (described below) that is in-frame with the Ctgf 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. 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 Ctgf-2A-dgCre-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed). In 2016, black male mice from generation N5 were sent to The Jackson Laboratory Repository. Upon arrival, males are used to cryopreserve sperm. To establish the living Ctgf-2A-dgCre-D mouse colony, an aliquot of the frozen sperm is used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

Allele

Symbol: Ccn2^{tm1.1(folA/cre)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5702703
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Ccn2
Marker name: cellular communication network factor 1
Chromosome: 10
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: Ccn2,cellular communication network factor 1,mouse, laboratory; GFP/cre,Green Fluorescent Protein/Cre recombinase fusion protein,
Site of expression: When induced, Cre recombinase activity is restricted to layer 6b of the cortex and in restricted populations within the cortical subplate of the brain.
Molecular note: The targeting vector contains, from 5' to 3', a partial exon 1 through intron 4 sequence containing an FRT3 site in intron 4, a partial exon 5 sequence up to (but not including) the endogenous stop codon, a T2A sequence that is in-frame with the coding sequence, a dgCre fusion gene (described below) that is in-frame with the 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 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. PhiC31 integrase-mediated recombination removed the AttB/AttP-flanked sequences. Trimethoprim-treatment is required to stabilize the cre protein. No EGFP is detected with or without induction.