Limits...
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

Schematics for Col1a1-directed expression of FRT-Cre-ERT2-FRT and FRT-STOP-FRT-Cre-ERT2, the removal of the neomycin selection cassette, and Southern blot for successful homologous recombination. Schematic of the Col1a1FRT-Cre-ER-T2-FRT (A) and Col1a1FRT-STOP-FRT-Cre-ER-T2 (B) alleles. The neomycin cassette is used for selection of ES cells with integration of the targeting construct. PhiC31 integrase is used to remove the neomycin selection cassette in vivo after germline transmission. Locations of genotyping primers are indicated on each schematic. (C) Southern blot analysis indicates successful homologous recombination of the Col1a1FRT-Cre-ER-T2-FRT-NEO allele in the E2, E1 and B7 clones. The B7 clone was used to generate the Col1a1FRT-Cre-ER-T2-FRT-NEO line. (D) Southern blot analysis indicates successful homologous recombination of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele in the E1, C1 and B11 clones. The B11 clone was used to generate the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4582108&req=5

DMM021204F2: Schematics for Col1a1-directed expression of FRT-Cre-ERT2-FRT and FRT-STOP-FRT-Cre-ERT2, the removal of the neomycin selection cassette, and Southern blot for successful homologous recombination. Schematic of the Col1a1FRT-Cre-ER-T2-FRT (A) and Col1a1FRT-STOP-FRT-Cre-ER-T2 (B) alleles. The neomycin cassette is used for selection of ES cells with integration of the targeting construct. PhiC31 integrase is used to remove the neomycin selection cassette in vivo after germline transmission. Locations of genotyping primers are indicated on each schematic. (C) Southern blot analysis indicates successful homologous recombination of the Col1a1FRT-Cre-ER-T2-FRT-NEO allele in the E2, E1 and B7 clones. The B7 clone was used to generate the Col1a1FRT-Cre-ER-T2-FRT-NEO line. (D) Southern blot analysis indicates successful homologous recombination of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele in the E1, C1 and B11 clones. The B11 clone was used to generate the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO line.

Mentions: To generate mice in which the expression of Cre-ERT2 is regulated by the Flp-FRT recombinase system and to fully utilize the large number of Cre-loxP-regulated transgenic alleles in the Rosa26 locus, such as fluorescent reporters and mutant genes, the two novel Cre-ERT2 alleles were targeted into the endogenous Col1a1 locus for ubiquitous expression (Fig. 2A,B). Targeting constructs for the generation of Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice consisted of the sequence from the Col1a1 genomic DNA, CAG promoter, Cre-ERT2 regulated by FRT sites, and a neomycin selection cassette flanked by attP and attB sites. Therefore, transcription of the targeted Cre-ERT2 recombinase will be driven from the endogenous Col1a1 locus and enhanced by the addition of a CAG promoter. Constructs of the two Cre-ERT2 alleles were electroporated into 129/SVJae embryonic stem (ES) cells and successfully targeted ES cells were selected by neomycin (G418) treatment. Positively selected ES cells were analyzed for successful homologous recombination by Southern blot of genomic DNA (Fig. 2C,D). Correctly targeted ES clones were injected into C57BL/6 blastocysts and male high-percentage chimeras were selected to breed with C57BL/6 females to identify germline transmission of the Cre-ERT2 alleles. The neomycin selection cassettes were removed by PhiC31-integrase-mediated recombination of the attP and attB sites in vivo by crossing the mice to the pre-existing Rosa26PhiC31 strain (Raymond and Soriano, 2007).Fig. 2.


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)

Schematics for Col1a1-directed expression of FRT-Cre-ERT2-FRT and FRT-STOP-FRT-Cre-ERT2, the removal of the neomycin selection cassette, and Southern blot for successful homologous recombination. Schematic of the Col1a1FRT-Cre-ER-T2-FRT (A) and Col1a1FRT-STOP-FRT-Cre-ER-T2 (B) alleles. The neomycin cassette is used for selection of ES cells with integration of the targeting construct. PhiC31 integrase is used to remove the neomycin selection cassette in vivo after germline transmission. Locations of genotyping primers are indicated on each schematic. (C) Southern blot analysis indicates successful homologous recombination of the Col1a1FRT-Cre-ER-T2-FRT-NEO allele in the E2, E1 and B7 clones. The B7 clone was used to generate the Col1a1FRT-Cre-ER-T2-FRT-NEO line. (D) Southern blot analysis indicates successful homologous recombination of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele in the E1, C1 and B11 clones. The B11 clone was used to generate the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

DMM021204F2: Schematics for Col1a1-directed expression of FRT-Cre-ERT2-FRT and FRT-STOP-FRT-Cre-ERT2, the removal of the neomycin selection cassette, and Southern blot for successful homologous recombination. Schematic of the Col1a1FRT-Cre-ER-T2-FRT (A) and Col1a1FRT-STOP-FRT-Cre-ER-T2 (B) alleles. The neomycin cassette is used for selection of ES cells with integration of the targeting construct. PhiC31 integrase is used to remove the neomycin selection cassette in vivo after germline transmission. Locations of genotyping primers are indicated on each schematic. (C) Southern blot analysis indicates successful homologous recombination of the Col1a1FRT-Cre-ER-T2-FRT-NEO allele in the E2, E1 and B7 clones. The B7 clone was used to generate the Col1a1FRT-Cre-ER-T2-FRT-NEO line. (D) Southern blot analysis indicates successful homologous recombination of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele in the E1, C1 and B11 clones. The B11 clone was used to generate the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO line.
Mentions: To generate mice in which the expression of Cre-ERT2 is regulated by the Flp-FRT recombinase system and to fully utilize the large number of Cre-loxP-regulated transgenic alleles in the Rosa26 locus, such as fluorescent reporters and mutant genes, the two novel Cre-ERT2 alleles were targeted into the endogenous Col1a1 locus for ubiquitous expression (Fig. 2A,B). Targeting constructs for the generation of Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice consisted of the sequence from the Col1a1 genomic DNA, CAG promoter, Cre-ERT2 regulated by FRT sites, and a neomycin selection cassette flanked by attP and attB sites. Therefore, transcription of the targeted Cre-ERT2 recombinase will be driven from the endogenous Col1a1 locus and enhanced by the addition of a CAG promoter. Constructs of the two Cre-ERT2 alleles were electroporated into 129/SVJae embryonic stem (ES) cells and successfully targeted ES cells were selected by neomycin (G418) treatment. Positively selected ES cells were analyzed for successful homologous recombination by Southern blot of genomic DNA (Fig. 2C,D). Correctly targeted ES clones were injected into C57BL/6 blastocysts and male high-percentage chimeras were selected to breed with C57BL/6 females to identify germline transmission of the Cre-ERT2 alleles. The neomycin selection cassettes were removed by PhiC31-integrase-mediated recombination of the attP and attB sites in vivo by crossing the mice to the pre-existing Rosa26PhiC31 strain (Raymond and Soriano, 2007).Fig. 2.

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