Identification of genes responsible for developmental mutant mice

转基因技术对于描述任何感兴趣的基因在体内的功能是非常有用和成功的。然而，传统的转基因技术存在一些技术限制。由于在构建过程中省略了内含子和正确体内表达所需的必要调控序列，因此转基因表达有时与相应内源基因的表达不同。所谓的“位置效应”影响外源基因的表达取决于其染色体整合位点，也保证了转基因的表达。此外，由于在噬菌体或cosmid载体上克隆的插入长度的限制，转基因的最大大小仅为40-50 kb。这些限制可以通过在转基因小鼠的产生中使用容纳亚megabase DNA的克隆系统来克服，例如YAC（酵母人工染色体）或BAC（细菌人工染色体）。另一方面，在过去的一个世纪里，已经有超过一千种小鼠突变体被发现和保存，并且在不久的将来，通过在国际老鼠群体中进行的几个“大规模”诱变项目，这一数字将急剧增加。然而，分离任何这些突变的负责基因的任务仍然是劳动密集型的，因此限制了这些实验资源的有效性。再一次，将大尺寸DNA引入小鼠生殖系的能力应该有助于纠正这种情况。通过将YACs或BACs引入突变小鼠并检查突变表型的功能挽救，应该可以确定大DNA片段上负责基因的位置。为此，我们建立了一系列将大尺寸DNA引入小鼠生殖系的技术。在这些过程中，我们发现YAC克隆含有嵌合和缺失等克隆产物，我们改用含有较少DNA重排的BAC。我们已经生产了50多只创始人转基因小鼠，这些小鼠携带的BAC克隆来自于Chr. 17的t复合体区域，尾巴扭结（tk）位点和PaxI位点。我们已经确定并表征了qkL基因，一个经典的神经突变候选基因，颤抖。我们引入了一个含有160 kb BAC的转基因品系，其中包含了整个qkI位点，并将该品系与突变动物杂交。震颤是一种由中枢神经系统髓鞘发育低下引起的震颤的隐性突变。引入含有BAC的qkl成功地纠正了突变表型；BAC纯合突变体未表现出震颤表型，髓鞘形成正常发生。我们还挽救了影响脊索发育和尾巴形态的短腺突变。这些结果证明了BAC转基因的有效性，特别是在小鼠突变基因的鉴定和功能分析方面。少

Transgenic techonology has been very useful and successful for delineating functions of any genes of interest in vivo. However, there are several technical limitations in conventional transgenic techniques. Since introns and essential regulatory sequences required for correct in vivo expression are omitted in the constructs, transgene expression is sometimes different from the expression of corresponding endogenous gene. So-called 'postion-effect' that affect foreign gene expression depending on its chromosomal integration site also conpromise the transgene expression. In addition, the maximum size of transgene is only 40-50 kb, due to constraints on the insert length that can be cloned in phage or cosmid vectors. These limitations can be overcome by using cloning systems which accomodate submegabase DNA such as YAC (yeast artificial chromosome) or BAC (bacterial artificial chromosome) in the generation of transgenic mice.On the other hand, more than a thousand mouse mutants have been … More identified and maintained over the past century and the number will drastically increase in near future by several 'large scale' mutagenesis projects operating in international mouse communities. However, the task of isolating the responsible gene for any of these mutantions is still labor intensive and so limits the usufullness of thisexperimental resources. Again, the ability to introduce large size DNA into the mouse germ lines should help rectify this situation. It should be possible to define the location of the responsible gene on a large DNA fragment by introducing YACs or BACs into mutant mice and examining the functional rescue of the mutant phenotype.Toward this end, we have established a series of techniques to introduce large size DNA into mouse germ lines. During these processes, we found that YAC clones contain cloning artifacts including chimerism and deletions, we have switched to use BAC which contain less DNA rearrangements.We have produced more than fifty founder transgenic mice carrying BAC clones derived from the t-complex region of Chr. 17, tail kinks (tk) locus and PaxI locus. We have identified and characterized qkL gene, a candidate for the classical neurological mutation, quaking. We introduced a transgenic line harboring a 160-kb BAC that contains the whole qkI locus and crossed the strain with the mutant animals. quaking is a recessive mutantion with tremors due to hypomyelination in CNS.Introduction of the qkl containing BAC successfully corrected the mutant phenotype ; homozygous mutant with the BAC do not show tremor phenotype and myelination occurred normaly. We have also rescued brachynry mutation which affect notochord development and tail morphology. These results thus demonstrated the usufullness of the BAC transgenesis particulary in identification and functional analysis of mutated genes in mice. Less