PRODUCTION OF MUTANT MICE AND ESTABLISHMENT OF EMBRYO BANK

利用化学诱变剂或x射线进行种系诱变，可以有效地生产携带突变的小鼠。然而，由于需要大量的动物住房资源，它们的应用受到限制。此外，还需要定位克隆方法来识别这些突变。另一种方法是胚胎干细胞诱变。自1989年以来，当第一批小鼠在胚胎干细胞中通过同源重组获得特异性修饰的基因座时，这些技术已用于突变约1%的小鼠基因座总数。然而，同源重组是费时费力的。一种更吸引人的方法是利用基因诱捕结构对胚胎干细胞进行随机插入诱变。为了进行基因陷阱实验，我们开发了一种新的筛选系统，将胚胎干细胞在悬浮培养中分化为胚状体（EB）。我们发现EB发育过程中的内胚层基因表达模式反映了小鼠体内发育过程中的顺序。因此，我们利用EB形成来寻找新的基因。我们还利用噬菌体P1的Cre-lox重组系统开发了一种新的位点特异性整合系统。lox位点由一个不对称的8 bp间隔序列组成，两侧是13 bp的反向重复序列。我们引入了左侧13bp元件（LE突变体lox位点）或右侧13bp元件（RE突变体lox位点）的核苷酸变化。LE突变体lox位点与RE突变体lox位点之间的重组产生了Cre难以识别的野生型和LE+RE突变位点，从而实现了稳定的整合。将该系统应用于基因诱捕，分离并测定了10个诱捕克隆的DNA序列。其中4个是已知基因，4个与ESTs同源，2个是未知基因，但与ESTs序列同源。因此，我们的基因陷阱系统在识别新基因和产生突变小鼠方面是非常有效的。

The efficient production of mice carrying mutations is possible by use of germline mutagenesis with chemical mutagents or X-rays. However, their application is limited, because the vast animal housing resource is required.Moreover, the positional cloning methods are required to identify these mutations. An alternative approach is mutagenesis in embryonic stem (ES) cell. Since 1989, when the first mice were derived with a locus specifically modified by homologous recombination in ES cells, these techniques have been used to mutate approximatery 1% of the total predicted number of mouse loci. However, homologous recombination is laborious and time-consuming. A more attracting approach is random insertional mutagenesis in ES cells using gene trap construct. To carry out the gene trap experiments, we developed a new screening system in which ES cells are differentiated into embryoid bodies (EB) in suspension culture. We found that the patterns of endoderm gene expression during EB development reflect the order found during mouse development in vivo. Thus, we used EB formation to search new genes. We also developed a new site-specific integration system using the Cre-lox recombination system of bacteriophage P1. The lox site is composed of an asymmetric 8 bp spacer flanked by 13 bp inverted repeats. We introduced nucleotide changes in the left 13 bp element (LE mutant lox site) or the right 13 bp element (RE mutant lox site). Recombination between the LE mutant lox site and RE mutant lox site produces a wild-type and a LE+RE mutant site that is poorly recognized by Cre, resulting in stable integration. By applying these new system to the gene trap, we isolated and determined the DNA sequences from 10 trap clones. Four of these are known genes, four are homolougous to ESTs, and two are unknown genes, but have sequence homology to ESTs. Thus, our gene trap system is quite efficient for identifying new genes and producing mutant mice.