Rapid generation of inducible mouse mutants

Abstract

We have generated an optimized inducible recombination system for conditional gene targeting based on a Cre recombinase-steroid receptor fusion. This configuration allows efficient Cre-mediated recombination in most organs of the mouse upon induction, without detectable background activity. An ES cell line, was established that carries the inducible recombinase and a loxP-flanked lacZ reporter gene. Out of this line, completely ES cell-derived mice were efficiently produced through tetraploid blastocyst complementation, without the requirement of mouse breeding. Our findings provide a new concept allowing the generation of inducible mouse mutants within 6 months, as compared to 14 months using the current protocol.

Figure 1

Generation of targeted ES cells. (A) Scheme of the gene targeting strategy. Both constructs were sequentially inserted into the Rosa26 locus by homologous recombination as depicted. E, EcoRV; X, XbaI; Bg, BglI. (B) Southern blot analysis of genomic DNA from ES cells containing the targeted insertion of CreER^T2 before and after FLP-mediated deletion of the neomycin resistance gene. The DNA of ES cells was digested with BglI and hybridized with probe 2. The sizes of the wild-type, targeted and Δneo alleles are 6.6, 7.6 and 9.0 kb, respectively. (C) Southern blot analysis of genomic DNA extracted from double-targeted ES cells carrying the rosa(CreER^T2) and rosa(lacZ reporter) alleles. Homologous recombination at the Rosa26 locus is detectable using EcoRV-digested genomic DNA and probe 1, resulting in an 11.7 kb band for the wild-type and a 2.5 kb band for the targeted allele. (D) Western blot analysis of CreER^T2 expression in targeted ES cells before and after neo deletion. The western blot was probed with a Cre antiserum as described in Materials and Methods. (E) Southern blot analysis of inducible recombination of the rosa(lacZ reporter) allele. Cells were treated for 4.5 days with 500 nM 4-hydroxytamoxifen (4-OHT). Southern blot analysis of EcoRV-digested DNA was performed using the rosa26-specific probe 1 depicted in (A). (F) Titration of 4-OHT inducible recombination. Cells were treated for 3 days with increasing concentrations of 4-OHT as indicated. Southern blot analysis of EcoRV-digested DNA was performed using the rosa26-specific probe 1.

Figure 2

Production efficiency of completely ES cell-derived mice. (A) ES cells with the indicated genotype were injected into tetraploid blastocysts. The percentages of blastocysts that developed into live born ES pups are depicted. (B) Southern blot analysis of DNA extracted from liver of ES mice (lanes 2–9). Genomic tail DNA from a wild-type ES mouse is used as a control (lane 1). DNA was digested with EcoRV and probed with the Rosa26-specific probe 1 as depicted in Figure 1A.

Figure 3

Inducible recombination in mice. Mice carrying different loxP-flanked alleles were fed daily with 5 mg tamoxifen (A–D) or 1 mg tamoxifen (E) for 5 days. Cre-mediated deletion of the loxP-flanked region was detected by Southern analysis 3 days after the treatment. (A) Rosa(CreER^T2/lacZ reporter) mice. Genomic DNA from various organs was digested with EcoRV and hybridized with a lacZ-specific probe. The percentages of deletion of the loxP-flanked allele, as quantified with a Bio-Imaging Analyzer, are indicated. (B) polβ^flox/rosa(CreER^T2) mice. Cre-mediated excision of the 1.5 kb loxP-flanked region was detected by Southern analysis. Genomic DNA from various organs was digested with BamHI and hybridized with a probe specific for the polymerase β gene (4). (C) ect2^flox/rosa(CreER^T2) mice. The loxP sites are 2.5 kb apart from each other flanking a FRT-flanked neomycin selection marker and exon 6 of the ect2 gene (described in Materials and Methods). Genomic DNA was digested with HpaI and hybridized with an ect2-specific probe hybridizing upstream of the loxP-flanked region. (D) ect2^floxΔ^neo/rosa(CreER^T2) mice. The ect2^floxΔ^neo allele was generated through FLP-mediated deletion of the neomycin selection marker from the ect2^flox allele resulting in a loxP-flanked region of 0.5 kb containing exon 6 of the ect2 gene (described in Materials and Methods). Genomic DNA was digested with HpaI and hybridized with an ect2-specific probe. (E) ect2^flox/rosa(CreER^T2) mice treated with 1 mg tamoxifen for 5 days. Genomic DNA was digested with HpaI and hybridized with an ect2-specific probe hybridizing upstream of the loxP-flanked region. Li, liver; Sp, spleen; Ki, kidney; He, heart; Lu, lung; Th, thymus; Mu, muscle; Si, small intestine; Br, brain; Te, testis; Co, colon.

Figure 4

Western blot analysis of CreER^T2 expression in ES mice. Proteins were extracted from rosa(CreER^T2) targeted ES mice and analyzed as described in Materials and Methods. Protein extracts from wild-type and rosa(CreER^T2) targeted ES cells are used as controls. The positions of bands representing CreER^T2 and actin are indicated. Li, liver; SP, spleen; Ki, kidney; He, heart; Lu, lung; Th, thymus; Mu, muscle; Si, small intestine; Br, brain; Fa, fat tissue; Te, testis.

Figure 5

The generation of inducible mouse mutants via the current protocol and via tetraploid blastocyst complementation. (A) The current protocol includes the generation of targeted ES cells, the FLP-mediated deletion of the neo gene, the production of chimeric mice, the breeding for germline transmission and another two breeding steps to combine two copies of the loxP-flanked allele with the recombinase transgene. Following this scheme, the generation of conditional mouse mutants usually takes 14 months. Chimeras can be directly bred to Cre transgenic mice supposing that appropriate Cre strains on pure genetic backgrounds are available that allow the detection of germline transmission by coat color. This would save 3 months required for one breeding step. (B) The new approach uses hybrid ES cells carrying the Cre gene with an appropriate expression pattern for conditional gene inactivation in mice. Following the introduction of loxP sites into both alleles of the target gene, the FLP-mediated deletion of the selection marker genes and the injection of these ES cells into tetraploid blastocysts, conditional mouse mutants are obtained within 6 month. The time required for each step is indicated.