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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 primary sarcomas generated in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice. Primary sarcomas were generated by intramuscular injection of adenovirus expressing FlpO recombinase into the hindlimb of 6-week-old Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice. (A) Primary sarcoma cells were isolated by dissociation of the bulk tumor and subsequent in vitro passage. The excision of the STOP cassette in the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele was confirmed via PCR for two tumors using primers FWD4 and REV2. Tail DNA from untreated Col1a1FRT-Cre-ER-T2-FRT mice serves as positive control for the absence of the STOP cassette, whereas the tail DNA from Col1a1FRT-STOP-FRT-Cre-ER-T2 mice serves as negative control for the retention of the STOP cassette. (B) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), one dose of tamoxifen was delivered by intraperitoneal injection. Tumors were collected 10 days after tamoxifen delivery. Immunofluorescence of tumor tissue showed no eGFP expression (iii), with the whole tumor retaining tdTomato (iv). (C) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), 4-hydroxytamoxifen was delivered by intramuscular injection at the site of the tumor. Tumors were collected 10 days after 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed scattered tdTomato expression labeling tumor vasculature and other stromal cells (iv), with the majority of tumor tissue expressing eGFP, which labels tumor parenchyma (iii). (D) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), one dose of 4-hydroxytamoxifen was delivered by subcutaneous injection between the scapula. Tumors were collected 24 h after 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed widespread tdTomato expression (iv), with small clusters of cells expressing eGFP, which labels tumor parenchyma (iii). (E) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=1), 4-hydroxytamoxifen was delivered by one dose of intraperitoneal injection, followed by three doses of subcutaneous injection between the scapula. Doses of 4-hydroxytamoxifen were given in 24 h intervals. Tumors were collected 1 day after the last dose of 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed scattered tdTomato expression labeling tumor vasculature and other stromal cells (iv), and cells with varying degrees of eGFP expression (iii). These results indicate that the Col1a1FRT-STOP-FRT-Cre-ER-T2 is functional and can be utilized for sequential mutagenesis in vivo. Scale bars: 100 µm.
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DMM021204F7: Characterization of primary sarcomas generated in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice. Primary sarcomas were generated by intramuscular injection of adenovirus expressing FlpO recombinase into the hindlimb of 6-week-old Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice. (A) Primary sarcoma cells were isolated by dissociation of the bulk tumor and subsequent in vitro passage. The excision of the STOP cassette in the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele was confirmed via PCR for two tumors using primers FWD4 and REV2. Tail DNA from untreated Col1a1FRT-Cre-ER-T2-FRT mice serves as positive control for the absence of the STOP cassette, whereas the tail DNA from Col1a1FRT-STOP-FRT-Cre-ER-T2 mice serves as negative control for the retention of the STOP cassette. (B) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), one dose of tamoxifen was delivered by intraperitoneal injection. Tumors were collected 10 days after tamoxifen delivery. Immunofluorescence of tumor tissue showed no eGFP expression (iii), with the whole tumor retaining tdTomato (iv). (C) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), 4-hydroxytamoxifen was delivered by intramuscular injection at the site of the tumor. Tumors were collected 10 days after 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed scattered tdTomato expression labeling tumor vasculature and other stromal cells (iv), with the majority of tumor tissue expressing eGFP, which labels tumor parenchyma (iii). (D) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), one dose of 4-hydroxytamoxifen was delivered by subcutaneous injection between the scapula. Tumors were collected 24 h after 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed widespread tdTomato expression (iv), with small clusters of cells expressing eGFP, which labels tumor parenchyma (iii). (E) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=1), 4-hydroxytamoxifen was delivered by one dose of intraperitoneal injection, followed by three doses of subcutaneous injection between the scapula. Doses of 4-hydroxytamoxifen were given in 24 h intervals. Tumors were collected 1 day after the last dose of 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed scattered tdTomato expression labeling tumor vasculature and other stromal cells (iv), and cells with varying degrees of eGFP expression (iii). These results indicate that the Col1a1FRT-STOP-FRT-Cre-ER-T2 is functional and can be utilized for sequential mutagenesis in vivo. Scale bars: 100 µm.

Mentions: To determine whether the STOP cassette for the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele can be excised by Flp-mediated recombination in vivo, mice with the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele were crossed to KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT mice (Lee et al., 2012) to generate Col1a1FRT-STOP-FRT-Cre-ER-T2/+; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT mice. Adenovirus expressing mammalian optimized Flp recombinase (Adeno-FlpO) was injected into the hindlimb of these mice to generate primary soft-tissue sarcomas. Once sarcomas developed, the bulk tumor tissues were excised and dissociated into single-cell suspensions and cultured in vitro. Genomic DNA was isolated from these cells and the recombination of the STOP cassette was verified by PCR (Fig. 7A). Tail DNA taken at the time of genotyping from a Col1a1FRT-Cre-ER-T2-FRT mouse was used as a positive control for the absence of STOP cassette, and tail DNA taken at the time of genotyping from a Col1a1FRT-STOP-FRT-Cre-ER-T2 mouse was used as a negative control for the recombined STOP cassette.Fig. 7.


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 primary sarcomas generated in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice. Primary sarcomas were generated by intramuscular injection of adenovirus expressing FlpO recombinase into the hindlimb of 6-week-old Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice. (A) Primary sarcoma cells were isolated by dissociation of the bulk tumor and subsequent in vitro passage. The excision of the STOP cassette in the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele was confirmed via PCR for two tumors using primers FWD4 and REV2. Tail DNA from untreated Col1a1FRT-Cre-ER-T2-FRT mice serves as positive control for the absence of the STOP cassette, whereas the tail DNA from Col1a1FRT-STOP-FRT-Cre-ER-T2 mice serves as negative control for the retention of the STOP cassette. (B) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), one dose of tamoxifen was delivered by intraperitoneal injection. Tumors were collected 10 days after tamoxifen delivery. Immunofluorescence of tumor tissue showed no eGFP expression (iii), with the whole tumor retaining tdTomato (iv). (C) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), 4-hydroxytamoxifen was delivered by intramuscular injection at the site of the tumor. Tumors were collected 10 days after 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed scattered tdTomato expression labeling tumor vasculature and other stromal cells (iv), with the majority of tumor tissue expressing eGFP, which labels tumor parenchyma (iii). (D) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), one dose of 4-hydroxytamoxifen was delivered by subcutaneous injection between the scapula. Tumors were collected 24 h after 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed widespread tdTomato expression (iv), with small clusters of cells expressing eGFP, which labels tumor parenchyma (iii). (E) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=1), 4-hydroxytamoxifen was delivered by one dose of intraperitoneal injection, followed by three doses of subcutaneous injection between the scapula. Doses of 4-hydroxytamoxifen were given in 24 h intervals. Tumors were collected 1 day after the last dose of 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed scattered tdTomato expression labeling tumor vasculature and other stromal cells (iv), and cells with varying degrees of eGFP expression (iii). These results indicate that the Col1a1FRT-STOP-FRT-Cre-ER-T2 is functional and can be utilized for sequential mutagenesis in vivo. Scale bars: 100 µm.
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DMM021204F7: Characterization of primary sarcomas generated in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice. Primary sarcomas were generated by intramuscular injection of adenovirus expressing FlpO recombinase into the hindlimb of 6-week-old Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice. (A) Primary sarcoma cells were isolated by dissociation of the bulk tumor and subsequent in vitro passage. The excision of the STOP cassette in the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele was confirmed via PCR for two tumors using primers FWD4 and REV2. Tail DNA from untreated Col1a1FRT-Cre-ER-T2-FRT mice serves as positive control for the absence of the STOP cassette, whereas the tail DNA from Col1a1FRT-STOP-FRT-Cre-ER-T2 mice serves as negative control for the retention of the STOP cassette. (B) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), one dose of tamoxifen was delivered by intraperitoneal injection. Tumors were collected 10 days after tamoxifen delivery. Immunofluorescence of tumor tissue showed no eGFP expression (iii), with the whole tumor retaining tdTomato (iv). (C) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), 4-hydroxytamoxifen was delivered by intramuscular injection at the site of the tumor. Tumors were collected 10 days after 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed scattered tdTomato expression labeling tumor vasculature and other stromal cells (iv), with the majority of tumor tissue expressing eGFP, which labels tumor parenchyma (iii). (D) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=2), one dose of 4-hydroxytamoxifen was delivered by subcutaneous injection between the scapula. Tumors were collected 24 h after 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed widespread tdTomato expression (iv), with small clusters of cells expressing eGFP, which labels tumor parenchyma (iii). (E) After tumor formation in Col1a1FRT-STOP-FRT-Cre-ER-T2; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT; Rosa26mTmG/+ mice (n=1), 4-hydroxytamoxifen was delivered by one dose of intraperitoneal injection, followed by three doses of subcutaneous injection between the scapula. Doses of 4-hydroxytamoxifen were given in 24 h intervals. Tumors were collected 1 day after the last dose of 4-hydroxytamoxifen delivery. Immunofluorescence of tumor tissue showed scattered tdTomato expression labeling tumor vasculature and other stromal cells (iv), and cells with varying degrees of eGFP expression (iii). These results indicate that the Col1a1FRT-STOP-FRT-Cre-ER-T2 is functional and can be utilized for sequential mutagenesis in vivo. Scale bars: 100 µm.
Mentions: To determine whether the STOP cassette for the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele can be excised by Flp-mediated recombination in vivo, mice with the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele were crossed to KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT mice (Lee et al., 2012) to generate Col1a1FRT-STOP-FRT-Cre-ER-T2/+; KrasFRT-STOP-FRT-G12D/+; p53FRT/FRT mice. Adenovirus expressing mammalian optimized Flp recombinase (Adeno-FlpO) was injected into the hindlimb of these mice to generate primary soft-tissue sarcomas. Once sarcomas developed, the bulk tumor tissues were excised and dissociated into single-cell suspensions and cultured in vitro. Genomic DNA was isolated from these cells and the recombination of the STOP cassette was verified by PCR (Fig. 7A). Tail DNA taken at the time of genotyping from a Col1a1FRT-Cre-ER-T2-FRT mouse was used as a positive control for the absence of STOP cassette, and tail DNA taken at the time of genotyping from a Col1a1FRT-STOP-FRT-Cre-ER-T2 mouse was used as a negative control for the recombined STOP cassette.Fig. 7.

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