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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

Sequential mutagenesis with Flp and Cre recombinases in Col1a1FRT-Cre-ER-T2-FRT and in Col1a1FRT-STOP-FRT-Cre-ER-T2 mice. (A) Col1a1FRT-Cre-ER-T2-FRT mice will express Cre-ERT2 recombinase ubiquitously from the Col1a1 locus. Exposure to Flp leads to excision of the Cre-ERT2 coding sequence flanked by FRT sites in the same cell and terminates Cre recombinase expression. Any other DNA flanked by FRT sites will be excised at the same time. Therefore, in these cells, subsequent tamoxifen exposure does not lead to Cre-mediated recombination of loxP sites. Without prior exposure to Flp, tamoxifen leads to Cre-ERT2 translocation into the nucleus and excision of loxP sites. (B) Col1a1FRT-STOP-FRT-Cre-ER-T2 mice do not express Cre-ERT2 at baseline. Cells exposed to Flp undergo excision of FRT sites including the STOP cassette upstream of Cre-ERT2, which results in transcription of Cre-ERT2. Subsequent tamoxifen exposure then leads to Cre-mediated recombination of loxP sites specifically in these cells. Cells without prior exposure to Flp do not recombine loxP sites when exposed to tamoxifen.
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DMM021204F1: Sequential mutagenesis with Flp and Cre recombinases in Col1a1FRT-Cre-ER-T2-FRT and in Col1a1FRT-STOP-FRT-Cre-ER-T2 mice. (A) Col1a1FRT-Cre-ER-T2-FRT mice will express Cre-ERT2 recombinase ubiquitously from the Col1a1 locus. Exposure to Flp leads to excision of the Cre-ERT2 coding sequence flanked by FRT sites in the same cell and terminates Cre recombinase expression. Any other DNA flanked by FRT sites will be excised at the same time. Therefore, in these cells, subsequent tamoxifen exposure does not lead to Cre-mediated recombination of loxP sites. Without prior exposure to Flp, tamoxifen leads to Cre-ERT2 translocation into the nucleus and excision of loxP sites. (B) Col1a1FRT-STOP-FRT-Cre-ER-T2 mice do not express Cre-ERT2 at baseline. Cells exposed to Flp undergo excision of FRT sites including the STOP cassette upstream of Cre-ERT2, which results in transcription of Cre-ERT2. Subsequent tamoxifen exposure then leads to Cre-mediated recombination of loxP sites specifically in these cells. Cells without prior exposure to Flp do not recombine loxP sites when exposed to tamoxifen.

Mentions: In order to develop technology for sequential mutagenesis in vivo using two site-specific recombinase systems, we generated Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice. The rationale to generate Col1a1FRT-Cre-ER-T2-FRT mice was to enable whole animal ubiquitous expression of Cre-ERT2 until exposure to Flp recombinase (Fig. 1A). After Flp-mediated recombination of the FRT sites, cells are no longer able to express Cre-ERT2 and therefore lose the ability to delete DNA flanked by loxP sites following exposure to tamoxifen. In this way, different mutations can be introduced in adjacent cells in vivo so that the consequences for intercellular interactions, such as those between cancer cells and stromal cells, can be studied. The rationale to generate Col1a1FRT-STOP-FRT-Cre-ER-T2 was that initially no cell expresses Cre-ERT2 because transcription of Cre-ERT2 is terminated by an upstream FRT-site-flanked transcription STOP cassette (Fig. 1B). However, after Flp-mediated recombination, the STOP cassette is excised. Therefore, these cells can initiate transcription of the Cre-ERT2 fusion protein, which, in response to subsequent exposure to tamoxifen, translocates into the nucleus to recombine DNA flanked by loxP sites. Cells without exposure to Flp will not be able to undergo Cre-mediated DNA recombination. In this way, the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele enables sequential mutations within the same cell over time. First, one mutation occurs in the cell from Flp recombinase, and then tamoxifen activates Cre recombinase in the same cell to mutate a second gene to study how the order of gene mutations affects cellular outcome. In addition, multiple genes might be mutated by Flp recombinase to initiate tumor development. Then, the role of a loxP-flanked gene in tumor maintenance can be studied because only the tumor cell will express Cre-ERT2. This allele can therefore be used to identify potential therapeutic targets.Fig. 1.


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)

Sequential mutagenesis with Flp and Cre recombinases in Col1a1FRT-Cre-ER-T2-FRT and in Col1a1FRT-STOP-FRT-Cre-ER-T2 mice. (A) Col1a1FRT-Cre-ER-T2-FRT mice will express Cre-ERT2 recombinase ubiquitously from the Col1a1 locus. Exposure to Flp leads to excision of the Cre-ERT2 coding sequence flanked by FRT sites in the same cell and terminates Cre recombinase expression. Any other DNA flanked by FRT sites will be excised at the same time. Therefore, in these cells, subsequent tamoxifen exposure does not lead to Cre-mediated recombination of loxP sites. Without prior exposure to Flp, tamoxifen leads to Cre-ERT2 translocation into the nucleus and excision of loxP sites. (B) Col1a1FRT-STOP-FRT-Cre-ER-T2 mice do not express Cre-ERT2 at baseline. Cells exposed to Flp undergo excision of FRT sites including the STOP cassette upstream of Cre-ERT2, which results in transcription of Cre-ERT2. Subsequent tamoxifen exposure then leads to Cre-mediated recombination of loxP sites specifically in these cells. Cells without prior exposure to Flp do not recombine loxP sites when exposed to tamoxifen.
© Copyright Policy - open-access
Related In: Results  -  Collection

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DMM021204F1: Sequential mutagenesis with Flp and Cre recombinases in Col1a1FRT-Cre-ER-T2-FRT and in Col1a1FRT-STOP-FRT-Cre-ER-T2 mice. (A) Col1a1FRT-Cre-ER-T2-FRT mice will express Cre-ERT2 recombinase ubiquitously from the Col1a1 locus. Exposure to Flp leads to excision of the Cre-ERT2 coding sequence flanked by FRT sites in the same cell and terminates Cre recombinase expression. Any other DNA flanked by FRT sites will be excised at the same time. Therefore, in these cells, subsequent tamoxifen exposure does not lead to Cre-mediated recombination of loxP sites. Without prior exposure to Flp, tamoxifen leads to Cre-ERT2 translocation into the nucleus and excision of loxP sites. (B) Col1a1FRT-STOP-FRT-Cre-ER-T2 mice do not express Cre-ERT2 at baseline. Cells exposed to Flp undergo excision of FRT sites including the STOP cassette upstream of Cre-ERT2, which results in transcription of Cre-ERT2. Subsequent tamoxifen exposure then leads to Cre-mediated recombination of loxP sites specifically in these cells. Cells without prior exposure to Flp do not recombine loxP sites when exposed to tamoxifen.
Mentions: In order to develop technology for sequential mutagenesis in vivo using two site-specific recombinase systems, we generated Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice. The rationale to generate Col1a1FRT-Cre-ER-T2-FRT mice was to enable whole animal ubiquitous expression of Cre-ERT2 until exposure to Flp recombinase (Fig. 1A). After Flp-mediated recombination of the FRT sites, cells are no longer able to express Cre-ERT2 and therefore lose the ability to delete DNA flanked by loxP sites following exposure to tamoxifen. In this way, different mutations can be introduced in adjacent cells in vivo so that the consequences for intercellular interactions, such as those between cancer cells and stromal cells, can be studied. The rationale to generate Col1a1FRT-STOP-FRT-Cre-ER-T2 was that initially no cell expresses Cre-ERT2 because transcription of Cre-ERT2 is terminated by an upstream FRT-site-flanked transcription STOP cassette (Fig. 1B). However, after Flp-mediated recombination, the STOP cassette is excised. Therefore, these cells can initiate transcription of the Cre-ERT2 fusion protein, which, in response to subsequent exposure to tamoxifen, translocates into the nucleus to recombine DNA flanked by loxP sites. Cells without exposure to Flp will not be able to undergo Cre-mediated DNA recombination. In this way, the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele enables sequential mutations within the same cell over time. First, one mutation occurs in the cell from Flp recombinase, and then tamoxifen activates Cre recombinase in the same cell to mutate a second gene to study how the order of gene mutations affects cellular outcome. In addition, multiple genes might be mutated by Flp recombinase to initiate tumor development. Then, the role of a loxP-flanked gene in tumor maintenance can be studied because only the tumor cell will express Cre-ERT2. This allele can therefore be used to identify potential therapeutic targets.Fig. 1.

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