IDENTIFICATION OF DEVELOPMENTAL CONTROL GENES USING YAC TRANSGENIC MICE

在这个项目中，我们试图建立一种结合位置克隆和转基因技术的实验方法来鉴定和分离突变基因。基因组分析技术的最新进展使我们能够将大型哺乳动物基因组DNA分离到YAC(酵母人工染色体)或BAC(细菌人工染色体)载体中。结合高效的遗传作图技术，可以对小鼠染色体上的任何突变进行定位，并将含有突变基因的区域缩小到亚百万碱基范围。然而，识别相关突变基因仍然是一项艰巨的任务，通过搜索突变型和野生型之间的DNA序列差异来识别基因的传统方法可能并不总是可行的。在这些情况下，利用转基因技术的另一种方法将有可能确定基因在大基因组dna上的位置，即引入含有候选基因…的基因组片段的yac或bac。更多地转化为突变小鼠，并为表型的挽救打分。我们现在正试图识别发育突变的突变基因，例如在小鼠17号染色体的T/t复合体中颤动的t^&lt；w5&gt；以及9号染色体上的tk(尾部扭结)。在每种情况下，我们都已经将突变基因所在的区域缩小到几百kb到1mb。为了寻找这些突变的突变基因，我们首先建立了携带YAC或BAC DNA的转基因小鼠的制备方法。制备YAC或BAC DNA的方法如下：在脉冲场凝胶上运行含有YAC或BAC DNA的琼脂糖插头，切下与YAC/BAC克隆相对应的条带，并用琼脂酶处理。酶消化后，释放的DNA用过滤装置洗涤和浓缩。将5 ng/mU的DNA溶液显微注射到小鼠受精卵中。当注入一个插入片段为650kb的YAC克隆时，我们可以建立三个分别携带-270kb、-380kb和-450kb的转基因株系。此外，我们还培育了两只转基因小鼠，发现两只小鼠都含有来自尾部扭结区域的完整的150kb BAC克隆。利用这些转基因小鼠，我们已经开始了育种实验，以询问这些转基因是否可以挽救突变的表型。较少

In this project, we try to establish an experimental approach coupling positional cloning and transgenic technologies for identification and isolation of mutated genes. Recent advances in genome analysis technologies enable us to isolate large size mammalian genomic DNA into YAC (Yeast artificial chromosome) or BAC (bacterial artificial chromosome) vectors. In combination with efficient genetic mapping techniques, it is feasible to map any mutation on mouse chromosome, and narrow down the region containing mutated gene to submega base range. However, identifying the relevant mutated gene can still be a formidable task, and a conventional method of identifying a gene by searching DNA sequence differences between mutant and wild-type may not always be possible. In those cases, alternative approach utilizing transgenic technologies would have potential to define the location of a gene on a large genomic DNA,i.e.introducing YAC or BACs containing genomic fragments harboring candidate genes … More into mutant mice and scoring for the rescue of the phenotype. We are now trying to identify mutated genes for developmental mutations such as t^<w5>, quaking both in the T/t complex of mouse Chromosome 17, and tk (tail kinks) on Chromosome 9. In each case, we have narrowed down the region where mutated gene resides to several hundred kb to 1 Mb. In order to find the mutated genes for these mutations, we first established the methodologies for production of transgenic mice carrying YAC or BAC DNA.YAC or BAC DNAs were prepared as follows ; an agarose plug containing YAC or BAC DNA was run on a pulsed field gel, and a band corresponding YAC/BAC clone waw excised and treated with agarase. After the enzyme digestion, released DNAs were washed and concentrated by a filtration apparatus. DNA solution of 5ng/mul was microinjected into mouse fertilized eggs. When a YAC clone with 650kb insert derived from the t^<w5> region was injected, we could establish three lines of transgenics carrying -270kb, -380kb and -450kb, respectively. Also, we have produced two transgenic mice, and found that both of mice contained intact, 150kb BAC clone derived from tail kinks region. Using these transgenic mice, we have begun breeding experiments to ask if these transgenes can rescue mutant phenotypes or not. Less