Early development will be studied in the mouse by the generation of developmental mutants. We will attempt to derive mutations both in unknown genes, using random retroviral infection or embryonic stem (ES) cells, as well as in two specific genes, c-myc and c-src, using ES cells in which these genes have been disrupted by retroviruses or homologous recombination. To facilitate the analysis of insertional mutations in transgenic mice, foreign DNA will be introduced into the germ line using retroviral infection. This method has been shown to produce insertional mutants at a similar frequency to that observed using microinjection of DNA into the zygote, but it offers the advantage over the latter method of producing insertions with no rearrangements of flanking sequences, which would otherwise complicate further analysis. The cloning of proviruses in mutant, strains will be facilitated by retroviral vectors containing a bacterial supF gene to allow selective cloning. A rapid screen of transgenic animals will be attempted by using retroviruses causing microphthalmia of the eye, or by PCR analysis of blood samples. The effect of parental imprinting on the transgene will also be investigated. To derive mutations in two specific genes, c-myc and c-src, ES cells will he infected repeatedly with retroviruses and pools of cells will be made. Cells in which the provirus has inserted within the gene will be identified using the polymerase chain reaction (PCR). These cells will be subcloned further prior to the formation of germ line chimeras. Alternatively, ES cells will be selected for homologous recombination into the genes using the pMCl or a promoterless neo selection system.
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