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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

Germline transmission, and successful PhiC- and FlpO-mediated recombination of attB/attP and FRT sites. (A) PCR of tail DNA for the neomycin cassette of the Col1a1FRT-Cre-ER-T2-FRT-NEO and wild-type Col1a1 locus indicate germline transmission of a single copy of the Col1a1FRT-Cre-ER-T2-FRT-NEOallele. (B) PCR for the recombined neomycin cassette (excised NEO), unrecombined neomycin cassette (NEO), wild-type Col1a1 locus and mutant PhiC31 allele indicate successful PhiC31-mediated deletion of the neomycin selection cassette in the Col1a1FRT-Cre-ER-T2-FRT-NEO allele, generating the Col1a1FRT-Cre-ER-T2-FRT allele. (C) PCR of tail DNA for the neomycin cassette of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO and wild-type Col1a1 locus indicate germline transmission of a single copy of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele. (D) PCR for the recombined neomycin cassette (excised NEO), unrecombined neomycin cassette (NEO), presence of STOP cassette, wild-type Col1a1 locus and mutant PhiC31 allele indicate successful PhiC31-mediated deletion of the neomycin selection cassette in the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele, generating the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele. Note that the amplified PCR product of the excised NEO band for the Col1a1FRT-Cre-ER-T2-FRTallele is slightly larger than that of the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele owing to the presence of the FRT site in between the FWD1 and REV1 primers (see Fig. 2 for schematics with the location of the FRT sites relative to the primers).
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DMM021204F3: Germline transmission, and successful PhiC- and FlpO-mediated recombination of attB/attP and FRT sites. (A) PCR of tail DNA for the neomycin cassette of the Col1a1FRT-Cre-ER-T2-FRT-NEO and wild-type Col1a1 locus indicate germline transmission of a single copy of the Col1a1FRT-Cre-ER-T2-FRT-NEOallele. (B) PCR for the recombined neomycin cassette (excised NEO), unrecombined neomycin cassette (NEO), wild-type Col1a1 locus and mutant PhiC31 allele indicate successful PhiC31-mediated deletion of the neomycin selection cassette in the Col1a1FRT-Cre-ER-T2-FRT-NEO allele, generating the Col1a1FRT-Cre-ER-T2-FRT allele. (C) PCR of tail DNA for the neomycin cassette of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO and wild-type Col1a1 locus indicate germline transmission of a single copy of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele. (D) PCR for the recombined neomycin cassette (excised NEO), unrecombined neomycin cassette (NEO), presence of STOP cassette, wild-type Col1a1 locus and mutant PhiC31 allele indicate successful PhiC31-mediated deletion of the neomycin selection cassette in the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele, generating the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele. Note that the amplified PCR product of the excised NEO band for the Col1a1FRT-Cre-ER-T2-FRTallele is slightly larger than that of the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele owing to the presence of the FRT site in between the FWD1 and REV1 primers (see Fig. 2 for schematics with the location of the FRT sites relative to the primers).

Mentions: Germline transmission of Col1a1FRT-Cre-ER-T2-FRT-NEO was verified by PCR for the construct-specific neomycin selection cassette on tail-tip DNA (Fig. 3A). Heterozygosity for the knock-in allele was demonstrated by PCR for the wild-type Col1a1 locus (Fig. 3A). Successful PhiC31-mediated excision of the neomycin selection cassette was verified by PCR for gene products specific to the recombined neomycin selection cassette (excised NEO), the unrecombined neomycin selection cassette (NEO), the wild-type Col1a1 locus and the mutant PhiC31 allele (Fig. 3B).Fig. 3.


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)

Germline transmission, and successful PhiC- and FlpO-mediated recombination of attB/attP and FRT sites. (A) PCR of tail DNA for the neomycin cassette of the Col1a1FRT-Cre-ER-T2-FRT-NEO and wild-type Col1a1 locus indicate germline transmission of a single copy of the Col1a1FRT-Cre-ER-T2-FRT-NEOallele. (B) PCR for the recombined neomycin cassette (excised NEO), unrecombined neomycin cassette (NEO), wild-type Col1a1 locus and mutant PhiC31 allele indicate successful PhiC31-mediated deletion of the neomycin selection cassette in the Col1a1FRT-Cre-ER-T2-FRT-NEO allele, generating the Col1a1FRT-Cre-ER-T2-FRT allele. (C) PCR of tail DNA for the neomycin cassette of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO and wild-type Col1a1 locus indicate germline transmission of a single copy of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele. (D) PCR for the recombined neomycin cassette (excised NEO), unrecombined neomycin cassette (NEO), presence of STOP cassette, wild-type Col1a1 locus and mutant PhiC31 allele indicate successful PhiC31-mediated deletion of the neomycin selection cassette in the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele, generating the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele. Note that the amplified PCR product of the excised NEO band for the Col1a1FRT-Cre-ER-T2-FRTallele is slightly larger than that of the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele owing to the presence of the FRT site in between the FWD1 and REV1 primers (see Fig. 2 for schematics with the location of the FRT sites relative to the primers).
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Related In: Results  -  Collection

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DMM021204F3: Germline transmission, and successful PhiC- and FlpO-mediated recombination of attB/attP and FRT sites. (A) PCR of tail DNA for the neomycin cassette of the Col1a1FRT-Cre-ER-T2-FRT-NEO and wild-type Col1a1 locus indicate germline transmission of a single copy of the Col1a1FRT-Cre-ER-T2-FRT-NEOallele. (B) PCR for the recombined neomycin cassette (excised NEO), unrecombined neomycin cassette (NEO), wild-type Col1a1 locus and mutant PhiC31 allele indicate successful PhiC31-mediated deletion of the neomycin selection cassette in the Col1a1FRT-Cre-ER-T2-FRT-NEO allele, generating the Col1a1FRT-Cre-ER-T2-FRT allele. (C) PCR of tail DNA for the neomycin cassette of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO and wild-type Col1a1 locus indicate germline transmission of a single copy of the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele. (D) PCR for the recombined neomycin cassette (excised NEO), unrecombined neomycin cassette (NEO), presence of STOP cassette, wild-type Col1a1 locus and mutant PhiC31 allele indicate successful PhiC31-mediated deletion of the neomycin selection cassette in the Col1a1FRT-STOP-FRT-Cre-ER-T2-NEO allele, generating the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele. Note that the amplified PCR product of the excised NEO band for the Col1a1FRT-Cre-ER-T2-FRTallele is slightly larger than that of the Col1a1FRT-STOP-FRT-Cre-ER-T2 allele owing to the presence of the FRT site in between the FWD1 and REV1 primers (see Fig. 2 for schematics with the location of the FRT sites relative to the primers).
Mentions: Germline transmission of Col1a1FRT-Cre-ER-T2-FRT-NEO was verified by PCR for the construct-specific neomycin selection cassette on tail-tip DNA (Fig. 3A). Heterozygosity for the knock-in allele was demonstrated by PCR for the wild-type Col1a1 locus (Fig. 3A). Successful PhiC31-mediated excision of the neomycin selection cassette was verified by PCR for gene products specific to the recombined neomycin selection cassette (excised NEO), the unrecombined neomycin selection cassette (NEO), the wild-type Col1a1 locus and the mutant PhiC31 allele (Fig. 3B).Fig. 3.

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