DEV02284 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Production of mice carrying the Jag1^flox conditional allele. (A) Targeting strategy. We generated Jag1^flox mice via homologous recombination in ES cells, in essentially the same way as described by Hozumi et al. (Hozumi et al., 2004) for production of Delta1^flox mice. The diagram shows the relevant part of the wild-type Jag1 gene, the targeting vector, the allele produced by homologous recombination and the alleles produced by Cre-mediated recombination. The targeting vector was designed to insert loxP sites to flank exons 4 and 5 of Jag1. Exon 4 (shaded grey) codes for the DSL domain, required for binding to Notch; Cre-mediated recombination is predicted to delete this and also introduce a frame shift that gives rise to a premature stop codon, yielding a functionally null allele. neo^r denotes neomycin-resistance gene. Arrows show primers used for genotyping by PCR. Filled arrowheads show loxP sites. (B) Southern blotting to check for recombination with the targeting vector. ES cells that had undergone homologous recombination were selected for neomycin resistance and identified by Southern blotting, For this, genomic DNA was digested with SpeI and hybridised with the probe indicated in A; homologous recombination eliminates the middle SpeI site, altering the set of digestion fragments. Clone 5G3 had a homologous recombinant allele (giving a 13.0 kb fragment, left lane) in addition to a copy of the wild-type gene, in contrast to a clone with only the endogenous allele of Jag1 (3.5 kb, right lane). (C) Southern blotting to test for removal of the neomycin cassette. To remove the neomycin cassette, clone 5G3 was transiently transfected with a Cre expression vector pMC-Cre; (see Hozumi et al., 2004), and 5G3-4 and 5G3-5 were obtained as neomycin-sensitive clones. Their genomic DNA was digested with SpeI and analysed by Southern blotting with the probe shown in A. Clone 5G3-4 carried the floxed Jag1 allele, Jag1^flox, with neomycin cassette removed, (11.7 kb, left lane), whereas clone 5G3-5 contained the fully recombined null allele, Jag1^Dflox (7.3 kb, right lane). (D) PCR analysis of Jag1^flox ES cells. The genotypes of clones 5G3 and 5G3-4 were checked by PCR, to make sure they contained the intended floxable element. For this, samples of the cells were infected with adenovirus expressing either Cre or GFP (as a control). DNA was extracted and analysed by PCR, using primers a, b and c (see A). The sample of cells exposed to Cre-expressing virus (lanes marked Cre+) yielded a mixture of PCR products corresponding to recombined (Jag1^Dflox) and unrecombined (Jag1^flox) alleles. Cells exposed to control virus (lanes marked Cre-) yielded only a product corresponding to the unrecombined allele. Cells of clone 5G3-4 were injected into blastocysts in the usual way to produce founder mice from which we derived progeny carrying the Jag1^flox allele.