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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 tamoxifen-mediated Cre-ERT2 activity in Col1a1FRT-Cre-ER-T2-FRT mice. (A) Immunofluorescence of tissues from 6-week-old Col1a1FRT-Cre-ER-T2-FRT; Rosa26mTmG/+ mice (n=2) without tamoxifen treatment show widespread tdTomato with tissue-specific minimal to no eGFP expression, with the pancreas showing the most Cre-ERT2 leakiness (Avii). (B) Immunofluorescence of tissues collected 30 days after one dose of intraperitoneal tamoxifen in 6-month-old mice (n=3) show extensive Cre-mediated expression of eGFP and tissue-specific tdTomato degradation. Note that the brain (Bxxxii) has the highest amount of tdTomato retention as well as areas of low/no eGFP expression. Scale bars: 50 µm.
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DMM021204F4: Characterization of tamoxifen-mediated Cre-ERT2 activity in Col1a1FRT-Cre-ER-T2-FRT mice. (A) Immunofluorescence of tissues from 6-week-old Col1a1FRT-Cre-ER-T2-FRT; Rosa26mTmG/+ mice (n=2) without tamoxifen treatment show widespread tdTomato with tissue-specific minimal to no eGFP expression, with the pancreas showing the most Cre-ERT2 leakiness (Avii). (B) Immunofluorescence of tissues collected 30 days after one dose of intraperitoneal tamoxifen in 6-month-old mice (n=3) show extensive Cre-mediated expression of eGFP and tissue-specific tdTomato degradation. Note that the brain (Bxxxii) has the highest amount of tdTomato retention as well as areas of low/no eGFP expression. Scale bars: 50 µm.

Mentions: To examine Cre-mediated recombination at a cellular level, tissues from Col1a1FRT-Cre-ER-T2-FRT; Rosa26mTmG/+ mice were examined by immunofluorescence with and without tamoxifen treatment (Fig. 4). Without Cre-mediated recombination, the Rosa26mTmG allele transcribes only the tdTomato fluorescent protein. After Cre-mediated excision of tdTomato, eGFP is expressed from the same locus. Because these mice are heterozygous for the Rosa26mTmG allele, any expression of eGFP indicates Cre-mediated excision of tdTomato. In tissues taken from 6-week-old mice (n=2) without tamoxifen treatment (Fig. 4A), expression of tdTomato was widespread, and there was minimal tissue-specific eGFP expression. Tissues with some eGFP expression included the pancreas (Fig. 4Avii) and, to a lesser extent, a few skeletal muscle fibers (Fig. 4Aiii). When tissues from 6-month-old mice (n=3) obtained 30 days after treatment with a single dose of 75 mg tamoxifen/kg body weight in corn oil by intraperitoneal injection were examined (Fig. 4B), there was widespread eGFP expression and minimal tdTomato retention. The tissue with highest tdTomato retention was the brain (Fig. 4Bxxxii), where there was diffuse low tdTomato expression and regions of high eGFP expression (Fig. 4Bxxxi). This might reflect diffusion limitations of tamoxifen metabolites across the blood-brain barrier, or brain-specific expression from the Col1a1 locus, synthesis of functional eGFP protein and degradation of tdTomato. The lower expression of tdTomato in the brain tissue of Col1a1FRT-Cre-ER-T2-FRT; Rosa26mTmG/+ mice treated with tamoxifen as compared to untreated controls (Fig. 4Axxxii) suggests that the rate of degradation of tdTomato plays a greater role. Additionally, it seems that certain cell types do have higher tdTomato or higher eGFP expression than others, which could be due to the activity of the Rosa26 locus or perhaps a consequence of the way the tissue is affected by processing. For example, the endothelial cells of the brain (Fig. 4Axxxii) have higher tdTomato expression than the neurons. The glomeruli of the kidney (Fig. 4Axx) have higher tdTomato expression than the collecting ducts. Overall, the lung parenchyma seems to have lower tdTomato expression (Fig. 4Axii) than other tissues such as the brain (Fig. 4Axxxii).Fig. 4.


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 tamoxifen-mediated Cre-ERT2 activity in Col1a1FRT-Cre-ER-T2-FRT mice. (A) Immunofluorescence of tissues from 6-week-old Col1a1FRT-Cre-ER-T2-FRT; Rosa26mTmG/+ mice (n=2) without tamoxifen treatment show widespread tdTomato with tissue-specific minimal to no eGFP expression, with the pancreas showing the most Cre-ERT2 leakiness (Avii). (B) Immunofluorescence of tissues collected 30 days after one dose of intraperitoneal tamoxifen in 6-month-old mice (n=3) show extensive Cre-mediated expression of eGFP and tissue-specific tdTomato degradation. Note that the brain (Bxxxii) has the highest amount of tdTomato retention as well as areas of low/no eGFP expression. Scale bars: 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

DMM021204F4: Characterization of tamoxifen-mediated Cre-ERT2 activity in Col1a1FRT-Cre-ER-T2-FRT mice. (A) Immunofluorescence of tissues from 6-week-old Col1a1FRT-Cre-ER-T2-FRT; Rosa26mTmG/+ mice (n=2) without tamoxifen treatment show widespread tdTomato with tissue-specific minimal to no eGFP expression, with the pancreas showing the most Cre-ERT2 leakiness (Avii). (B) Immunofluorescence of tissues collected 30 days after one dose of intraperitoneal tamoxifen in 6-month-old mice (n=3) show extensive Cre-mediated expression of eGFP and tissue-specific tdTomato degradation. Note that the brain (Bxxxii) has the highest amount of tdTomato retention as well as areas of low/no eGFP expression. Scale bars: 50 µm.
Mentions: To examine Cre-mediated recombination at a cellular level, tissues from Col1a1FRT-Cre-ER-T2-FRT; Rosa26mTmG/+ mice were examined by immunofluorescence with and without tamoxifen treatment (Fig. 4). Without Cre-mediated recombination, the Rosa26mTmG allele transcribes only the tdTomato fluorescent protein. After Cre-mediated excision of tdTomato, eGFP is expressed from the same locus. Because these mice are heterozygous for the Rosa26mTmG allele, any expression of eGFP indicates Cre-mediated excision of tdTomato. In tissues taken from 6-week-old mice (n=2) without tamoxifen treatment (Fig. 4A), expression of tdTomato was widespread, and there was minimal tissue-specific eGFP expression. Tissues with some eGFP expression included the pancreas (Fig. 4Avii) and, to a lesser extent, a few skeletal muscle fibers (Fig. 4Aiii). When tissues from 6-month-old mice (n=3) obtained 30 days after treatment with a single dose of 75 mg tamoxifen/kg body weight in corn oil by intraperitoneal injection were examined (Fig. 4B), there was widespread eGFP expression and minimal tdTomato retention. The tissue with highest tdTomato retention was the brain (Fig. 4Bxxxii), where there was diffuse low tdTomato expression and regions of high eGFP expression (Fig. 4Bxxxi). This might reflect diffusion limitations of tamoxifen metabolites across the blood-brain barrier, or brain-specific expression from the Col1a1 locus, synthesis of functional eGFP protein and degradation of tdTomato. The lower expression of tdTomato in the brain tissue of Col1a1FRT-Cre-ER-T2-FRT; Rosa26mTmG/+ mice treated with tamoxifen as compared to untreated controls (Fig. 4Axxxii) suggests that the rate of degradation of tdTomato plays a greater role. Additionally, it seems that certain cell types do have higher tdTomato or higher eGFP expression than others, which could be due to the activity of the Rosa26 locus or perhaps a consequence of the way the tissue is affected by processing. For example, the endothelial cells of the brain (Fig. 4Axxxii) have higher tdTomato expression than the neurons. The glomeruli of the kidney (Fig. 4Axx) have higher tdTomato expression than the collecting ducts. Overall, the lung parenchyma seems to have lower tdTomato expression (Fig. 4Axii) than other tissues such as the brain (Fig. 4Axxxii).Fig. 4.

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