Identification of genes responsible for developmental mutant mice

转基因技术已经非常有用和成功地描绘任何感兴趣的基因在体内的功能。然而，在常规转基因技术中存在若干技术限制。由于在构建体中省略了正确体内表达所需的内含子和必需调控序列，因此转基因表达有时不同于相应内源基因的表达。所谓的“位置效应”，即外源基因的表达依赖于其染色体整合位点，也干扰了转基因的表达。此外，由于可以克隆到噬菌体或粘粒载体中的插入片段长度的限制，转基因的最大大小仅为40-50 kb。这些局限性可以通过在转基因小鼠的产生中使用容纳亚巨噬细胞DNA的克隆系统如YAC（酵母人工染色体）或BAC（细菌人工染色体）来克服。 ...更多信息 在过去的世纪中鉴定和维持，并且在不久的将来，通过在国际小鼠社区中操作的几个“大规模”诱变项目，数量将急剧增加。然而，分离这些突变的责任基因的任务仍然是劳动密集型的，因此限制了这种实验资源的使用。同样，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YAC或BAC导入突变小鼠并检测突变表型的功能拯救，可以确定大DNA片段上的责任基因的位置。为此，我们建立了一系列将大DNA导入小鼠生殖系的技术。在此过程中，我们发现YAC克隆中含有嵌合和缺失等克隆假象，我们改用DNA重排较少的BAC，并成功地制备了50多只携带来自Chr. 17的t复合体区、tk位点和PaxI位点的BAC克隆的founder转基因小鼠。我们已经确定和特点qkL基因，候选人的经典神经突变，震颤。我们引入了一个转基因株系，该株系含有一个160 kb的BAC，该BAC含有整个qkI基因座，并将该株系与突变动物杂交。quaking是一种隐性突变，由于CNS中髓鞘形成不足而引起震颤，引入含有BAC的qkl成功地纠正了突变表型，具有BAC的纯合突变体不显示震颤表型，髓鞘形成正常。我们还拯救了影响脊索发育和尾部形态的短雌突变。这些结果表明BAC转基因技术在小鼠突变基因的鉴定和功能分析中的应用。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