Many genes are upregulated during the progression of cutaneous malignant melanoma. To better understand the role of these genes, functional studies are needed. Our project aimed at developing a new strategy to produce mice carrying virtually any DNA sequence of interest inserted in place of the first exon of the Dct gene. Dct encodes dopachrome tautomerase, a melanogenic enzyme expressed in melanocytes and their precursors. Our goal was to increase the frequency of homologous recombination at the Dct locus in embryonic stem cells (ES) by inducing double-strand break (DSB) at this locus using the endonuclease properties of the yeast I-SceI meganuclease. We produced an ES cell line in which a single I-SceI recognition site was inserted into the first intron of the Dct gene. Our hypothesis was that inducing a double-strand break at the Dct locus would be recombinogenic, and that, in presence of a repair template, the frequency of homologous recombination would increase significantly. We tested this hypothesis and integrated successively the reporter genes, Lago1 and the H2B-mCherry, at the Dct locus. Our data indicate that, surprisingly, induction of DSB does not promote homologous recombination at the Dct locus. We analyzed the expression pattern of the reporter genes, Lago1 and the H2B-mCherry, inserted at the Dct locus in cultured cells, embryos or adults
Development of a tool for the genetic modification at the Dct locus in mouse embryonic stem cells using the meganuclease I-SceI