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The generation and characterization of novel Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice for sequential mutagenesis.

Zhang M, Kirsch DG - Dis Model Mech (2015)

Bottom Line: This application of dual recombinase technology can be used to dissect the role of stromal cells in tumor development and cancer therapy.To potentially utilize the large number of Cre-loxP-regulated transgenic alleles that have already been targeted into the Rosa26 locus, such as different reporters and mutant genes, we targeted the two novel Cre-ER(T2) alleles into the endogenous Col1a1 locus for ubiquitous expression.These two new novel mouse strains will be complementary to each other and will enable the exploration of complex biological questions in development, normal tissue homeostasis and cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27708, USA.

No MeSH data available.


Related in: MedlinePlus

Characterization of Col1a1FRT-STOP-FRT-Cre-ER-T2 mice. (A) Immunofluorescence of tissues from 6-month-old Col1a1FRT-STOP-FRT-Cre-ER-T2; Rosa26mTmG/+ mice (n=2) without tamoxifen treatment show widespread tdTomato expression and no eGFP expression. (B) Immunofluorescence of tissues collected 30 days after one dose of intraperitoneal tamoxifen in 6-month-old mice (n=2) continue to show widespread tdTomato expression and no eGFP expression, indicating that the STOP cassette prevents expression of Cre-ERT2 in the absence of FlpO-mediated recombination and excision of the STOP cassette. Scale bars: 50 µm.
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DMM021204F6: Characterization of Col1a1FRT-STOP-FRT-Cre-ER-T2 mice. (A) Immunofluorescence of tissues from 6-month-old Col1a1FRT-STOP-FRT-Cre-ER-T2; Rosa26mTmG/+ mice (n=2) without tamoxifen treatment show widespread tdTomato expression and no eGFP expression. (B) Immunofluorescence of tissues collected 30 days after one dose of intraperitoneal tamoxifen in 6-month-old mice (n=2) continue to show widespread tdTomato expression and no eGFP expression, indicating that the STOP cassette prevents expression of Cre-ERT2 in the absence of FlpO-mediated recombination and excision of the STOP cassette. Scale bars: 50 µm.

Mentions: To examine the leakiness of Cre-ERT2 in Col1a1FRT-STOP-FRT-Cre-ER-T2 mice, tissues from 6-month-old Col1a1FRT-STOP-FRT-Cre-ER-T2; Rosa26mTmG/+ mice were examined by immunofluorescence with (n=2) and without (n=2) tamoxifen treatment (Fig. 6). In the absence of tamoxifen, there was widespread tdTomato expression without evidence of eGFP expression in any of the tissues examined (Fig. 6A). When tissues were collected 30 days after intraperitoneal delivery of one dose of 75 mg tamoxifen/kg body weight in corn oil, there continued to be widespread tdTomato expression without evidence of eGFP expression in any of the tissues examined (Fig. 6B). These results indicate that the STOP cassette is functional and does not allow for Cre-ERT2-mediated transcription in the absence of Flp-mediated recombination and excision of the STOP cassette.Fig. 6.


The generation and characterization of novel Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice for sequential mutagenesis.

Zhang M, Kirsch DG - Dis Model Mech (2015)

Characterization of Col1a1FRT-STOP-FRT-Cre-ER-T2 mice. (A) Immunofluorescence of tissues from 6-month-old Col1a1FRT-STOP-FRT-Cre-ER-T2; Rosa26mTmG/+ mice (n=2) without tamoxifen treatment show widespread tdTomato expression and no eGFP expression. (B) Immunofluorescence of tissues collected 30 days after one dose of intraperitoneal tamoxifen in 6-month-old mice (n=2) continue to show widespread tdTomato expression and no eGFP expression, indicating that the STOP cassette prevents expression of Cre-ERT2 in the absence of FlpO-mediated recombination and excision of the STOP cassette. Scale bars: 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582108&req=5

DMM021204F6: Characterization of Col1a1FRT-STOP-FRT-Cre-ER-T2 mice. (A) Immunofluorescence of tissues from 6-month-old Col1a1FRT-STOP-FRT-Cre-ER-T2; Rosa26mTmG/+ mice (n=2) without tamoxifen treatment show widespread tdTomato expression and no eGFP expression. (B) Immunofluorescence of tissues collected 30 days after one dose of intraperitoneal tamoxifen in 6-month-old mice (n=2) continue to show widespread tdTomato expression and no eGFP expression, indicating that the STOP cassette prevents expression of Cre-ERT2 in the absence of FlpO-mediated recombination and excision of the STOP cassette. Scale bars: 50 µm.
Mentions: To examine the leakiness of Cre-ERT2 in Col1a1FRT-STOP-FRT-Cre-ER-T2 mice, tissues from 6-month-old Col1a1FRT-STOP-FRT-Cre-ER-T2; Rosa26mTmG/+ mice were examined by immunofluorescence with (n=2) and without (n=2) tamoxifen treatment (Fig. 6). In the absence of tamoxifen, there was widespread tdTomato expression without evidence of eGFP expression in any of the tissues examined (Fig. 6A). When tissues were collected 30 days after intraperitoneal delivery of one dose of 75 mg tamoxifen/kg body weight in corn oil, there continued to be widespread tdTomato expression without evidence of eGFP expression in any of the tissues examined (Fig. 6B). These results indicate that the STOP cassette is functional and does not allow for Cre-ERT2-mediated transcription in the absence of Flp-mediated recombination and excision of the STOP cassette.Fig. 6.

Bottom Line: This application of dual recombinase technology can be used to dissect the role of stromal cells in tumor development and cancer therapy.To potentially utilize the large number of Cre-loxP-regulated transgenic alleles that have already been targeted into the Rosa26 locus, such as different reporters and mutant genes, we targeted the two novel Cre-ER(T2) alleles into the endogenous Col1a1 locus for ubiquitous expression.These two new novel mouse strains will be complementary to each other and will enable the exploration of complex biological questions in development, normal tissue homeostasis and cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27708, USA.

No MeSH data available.


Related in: MedlinePlus