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.
Hongkui Zeng, Allen Institute for Brain Science
Genetic Background | Generation |
---|---|
|
Allele Type | Gene Symbol | Gene Name |
---|---|---|
Targeted (Recombinase-expressing, Inducible) | Ccn2 | cellular communication network factor 1 |
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 ecDHFRR12Y/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 ecDHFRR12Y/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 ecDHFRR12Y/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).
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 ecDHFRR12Y/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).
Expressed Gene | Ccn2, cellular communication network factor 1, mouse, laboratory |
---|---|
Expressed Gene | 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. |
Allele Name | targeted mutation 1.1, Hongkui Zeng |
---|---|
Allele Type | Targeted (Recombinase-expressing, Inducible) |
Allele Synonym(s) | Ctgf-2A-dgCre; Ctgf-2A-dgCre-D |
Gene Symbol and Name | Ccn2, cellular communication network factor 1 |
Gene Synonym(s) | |
Promoter | Ccn2, cellular communication network factor 1, mouse, laboratory |
Expressed Gene | Ccn2, cellular communication network factor 1, mouse, laboratory |
Expressed Gene | 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. |
Strain of Origin | (129S6/SvEvTac x C57BL/6NCrl)F1 |
Chromosome | 10 |
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. |
When maintaining a live colony, heterozygous mice may be bred together, to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). To date (December 2015), it has not been attempted to make this strain homozygous.
When using the Ctgf-2A-dgCre-D mouse strain in a publication, please cite the originating article(s) and include JAX stock #028535 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
---|---|---|
Heterozygous or wildtype for Ctgf<tm1.1(folA/cre)Hze> |
Frozen Mouse Embryo | B6.Cg-Ctgf<tm1.1(folA/cre)Hze>/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | B6.Cg-Ctgf<tm1.1(folA/cre)Hze>/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | B6.Cg-Ctgf<tm1.1(folA/cre)Hze>/J Frozen Embryo | $3373.50 |
Frozen Mouse Embryo | B6.Cg-Ctgf<tm1.1(folA/cre)Hze>/J Frozen Embryo | $3373.50 |
Terms are granted by individual review and stated on the customer invoice(s) and account statement. These transactions are payable in U.S. currency within the granted terms. Payment for services, products, shipping containers, and shipping costs that are rendered are expected within the payment terms indicated on the invoice or stated by contract. Invoices and account balances in arrears of stated terms may result in The Jackson Laboratory pursuing collection activities including but not limited to outside agencies and court filings.
The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.
What information were you hoping to find through your search?
How easy was it to find what you were looking for?
We may wish to follow up with you. Enter your email if you are happy for us to connect and reachout to you with more questions.
Please Enter a Valid Email Address
Thank you for sharing your feedback! We are working on improving the JAX Mice search. Come back soon for exciting changes.