We are using two approaches to identify and isolate genes involved in the regulated growth and differentiation of the mammalian embryo and fetus. The first of these is insertional mutagenesis, in which mutant phenotypes are generated by the insertion of exogenous DNA into a gene. This also creates a molecular tag at the mutant locus, providing direct access to the gene. For several years, we have analyzed transgenic mouse strains derived from embryonic stem (ES) cells deliberately infected in tissue culture with retroviruses to generate insertional mutations. We have screened over 40 proviral insertions specifically for mutations that lead to prenatal lethality, indicating that the gene is absolutely essential for normal development. This screen has identified 4 recessive prenatal lethal mutations tightly linked to, and presumably caused by, proviral insertion. Currently we are focussing on the 412-r mutation. We isolated the retroviral insertion site and surrounding DNA on a cosmid. Fragments of candidate genes were obtained using exon-trapping and extended by using 5 and 3 RACE, and by screening a testes cDNA library. The best candidate gene is one in which the retrovirus has integrated into a non-coding exon. This gene has multiple splicing isoforms, with the expression of one isoform missing in homozygous 412-r mutant embryos. Interestingly, some of the exons of this gene are found as testes specific 5 nontranslated exons of the gene for the muscle form of phosphofructokinase (PFK-M) which lies 25 kilobases downstream. The phenotypic analysis of the 412-r mutation has been delayed by the need to breed onto a different strain. This has been accomplished and detailed examination of mutant embryos now is taking place.The second approach to screen for developmental genes is based on the premise that many critical regulatory molecules are ubiquitinated during embryonic development, and can be identified by their interaction with ubiquitinating enzymes. For this screen we utilized one of the few well characterized ubiquitin ligases, Nedd-4 to carry out a yeast two-hybrid screen on a cDNA library from midgestation mouse embryos. This resulted in the isolation of 4 novel genes. Further in vitro experiments showed that 3 of these genes encode proteins that could bind to Nedd-4 and 2 could be ubiquitinated by Nedd-4. Followup studies using transfection of expression constructs into COS cells showed that protein levels for one of these novel genes is regulated by proteosome-mediated degradation. Co-transfection with a Nedd-4 expression vector greatly decreased half-life, suggesting that it may indeed be a true substrate for Nedd-4 ubiquitination in vivo. - Animal models, Development, embryonic stem cells, Genetics, in situ hybridization, Insertional Mutagenesis, Retroviruses, Transgenic Mice,
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