SCREENING FOR GENES ESSENTIAL FOR DEVELOPMENT OF THE MOUSE EMBRYO

我们正在使用两种方法来确定和分离基因参与调节生长和分化的哺乳动物胚胎和胎儿。第一种是插入诱变，其中通过将外源DNA插入到基因中产生突变表型。这也在突变位点产生了一个分子标签，提供了直接进入基因的途径。几年来，我们分析了转基因小鼠品系来自胚胎干细胞（ES）故意感染组织培养与逆转录病毒产生插入突变。我们已经筛选了超过40个前病毒插入，特别是导致产前致死性的突变，表明该基因是绝对必要的正常发育。该筛选鉴定出4种与前病毒插入紧密相关且可能由前病毒插入引起的隐性产前致死突变。目前，我们正在关注412-r突变。我们在粘粒上分离出逆转录病毒插入位点和周围的DNA。采用外显子捕获法获得候选基因片段，用5和3 RACE扩增，并筛选睾丸cDNA文库。最好的候选基因是其中逆转录病毒已整合到非编码外显子中的基因。该基因具有多种剪接异构体，其中一种异构体的表达在纯合412-r突变胚胎中缺失。有趣的是，该基因的一些外显子被发现是位于25个磷酸果糖激酶下游的肌肉形式磷酸果糖激酶（PFK-M）基因的睾丸特异性5个非翻译外显子。412-r突变的表型分析由于需要培育不同的菌株而被推迟。第二种筛选发育基因的方法是基于这样一个前提，即许多关键的调控分子在胚胎发育过程中被泛素化，并且可以通过它们与泛素化酶的相互作用来识别。对于这个屏幕，我们利用了一个为数不多的充分表征的泛素连接酶，Nedd-4进行酵母双杂交筛选的cDNA文库从妊娠中期小鼠胚胎。这导致了4个新基因的分离。进一步的体外实验表明，这些基因中有3个编码可以与Nedd-4结合的蛋白质，2个可以被Nedd-4泛素化。后续研究使用转染到COS细胞的表达构建体表明，这些新基因的蛋白质水平的调节蛋白质体介导的降解。与Nedd-4表达载体共转染大大降低了半衰期，表明它可能确实是体内Nedd-4泛素化的真正底物。- 动物模型，发育，胚胎干细胞，遗传学，原位杂交，插入突变，逆转录病毒，转基因小鼠，

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,