SCREENING FOR GENES ESSENTIAL FOR DEVELOPMENT OF THE MOUSE EMBRYO

我们正在使用两种方法来鉴定和分离参与哺乳动物胚胎和胎儿的调节生长和分化的基因。其中第一种是插入性突变，通过将外源DNA插入到基因中产生突变表型。这也在突变位点创建了一个分子标签，提供了对基因的直接访问。几年来，我们分析了来自胚胎干细胞（ES）的转基因小鼠品系，这些胚胎干细胞在组织培养中被逆转录病毒感染以产生插入突变。我们已经筛选了40多个前病毒插入，专门针对导致产前致死的突变，表明该基因对正常发育绝对必要。该筛查已确定了4种隐性产前致死突变，与前病毒插入密切相关，并且可能是由前病毒插入引起的。目前我们正在关注412-r突变。我们在一个宇宙中分离了逆转录病毒的插入位点和周围的DNA。候选基因片段通过外显子捕获获得，并通过5和3 RACE和筛选睾丸cDNA文库进行扩展。最佳候选基因是逆转录病毒整合到非编码外显子中的基因。该基因具有多个剪接异构体，在纯合子412-r突变胚胎中缺失一个异构体的表达。有趣的是，该基因的一些外显子被发现是位于下游25千碱基的磷酸果糖激酶（PFK-M）肌肉形式基因的睾丸特异性5个非翻译外显子。412-r突变的表型分析由于需要在不同的菌株上繁殖而被推迟。这已经完成，现在正在进行突变胚胎的详细检查。第二种筛选发育基因的方法是基于许多关键调控分子在胚胎发育过程中被泛素化的前提，并且可以通过它们与泛素化酶的相互作用来识别。在这个筛选中，我们利用了少数几个被充分表征的泛素连接酶之一，ned -4，对妊娠中期小鼠胚胎的cDNA文库进行酵母双杂交筛选。结果分离到4个新基因。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，2个可以被Nedd-4泛素化。后续研究将表达构建体转染到COS细胞中，发现其中一种新基因的蛋白水平受蛋白体介导的降解调节。与ned -4表达载体共转染大大缩短了半衰期，表明它可能确实是体内ned -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,