PRODUCTION OF MOUSE MODELS FOR MALFORMATION BY GENE TRAP

1979年，在细菌中首次描述了一种监测基因组转录活性区域的策略。它涉及将报告基因引入基因组，需要获得顺式作用的DNA序列来激活报告基因的表达。这种方法最近在高等生物中应用，使用适合真核生物转录单位的修饰载体。在小鼠中，通过逆转录病毒感染或DNA显微注射产生的转基因小鼠的插入突变鉴定了未知基因。然而，这种方法既费力又耗时。通过使用胚胎干细胞，增强子陷阱和基因陷阱现在在小鼠中成为可能。在诱捕载体中，细菌lacZ基因通常作为报告基因。然而，这种基因产物被认为对某些类型的细胞有毒性作用。我们发现b-半乳糖苷酶在细胞核中的靶表达降低了这种毒性。利用基因诱捕法，我们发现在大约60%的ES诱捕克隆中发现了新的基因，并且在大多数情况下，侧翼小鼠基因组的3'区被插入事件删除或重排。我们分离了6个在维甲酸诱导分化前表达lacZ的ES诱捕克隆。未知基因的启动子区域，称为Ayul，是从这些陷阱克隆之一分离出来的。该基因在成年小鼠的脑神经细胞、胃上皮细胞、十二指肠上皮细胞和睾丸中表达。b-半乳糖苷酶在8.5天胚胎中肠中也有活性，此时胃肠道开始形成，表明Ayul基因参与了胃肠道的形成。

A strategy to monitor transcriptionally active regions of the genome was first described in bacteria in 1979. It involves the introdution of reporter into the genome that require the acquisition of cis-acting DNA sequences to activate reporter gene expression. This approach has been applied more recently in higher organisms using modified vectors suitable for eukaryotic transcription units. In the mouse unknown genes have been identified by insertional mutation in transgenic mice generated by retroviral infection or DNA microinjection. However, this approach is laborious and time-consuming. Through the use of embryonic stem (ES) cells, the enhancer traps as well as the gene trap are now possible in the mouse. In the trap vector bacterial lacZ gene is usually used as a reporter gene. However, this gene product is thought to exert toxic effect on certain types of cell. We fpoud that this toxicity was reduced by the target expression of b-galactosidase in the nucleus. Using the gene trap method, we demonstrated that new genes were identified in about 60% ES trap clones, and that the 3'region of flanking mouse genome was deleted of rearranged by insertion event in most of cases. We isolated 6 ES trap clones that showed expression of lacZ before induction of differentiatio by retinoic acid. The promoter region of the unknown gene, termed Ayul, was isolated from one of these trap clones. This gene is expressed in the nerve cell of brain, epithelial cells of sstomach and duodenum, and testis of adult mouse. b-galactosidase activity was also detected in the midgut of 8.5 day embry when the gastro-intesitinal tract starts to form, suggesting that the Ayul gene is involved in the formation of gastro-intestinal tract.