IDENTIFICATION OF DISEASE GENE USING BAC TRANSGENIC MICE

在复制染色体时，染色体末端的DNA不能丢失，每一条染色体在细胞分裂时都要分离成子细胞。要完成这一过程，至少需要三个要素：复制起始点、端粒和着丝粒。在酵母和大肠杆菌中，这些元素已经被很好地表征并克隆出来。利用这些元素，现在可以构建人工染色体，如酵母人工染色体（YAC）或细菌人工染色体（BAC）。这些人工染色体可用于分离BAC中长达几百kb的DNA大片段和YAC中长达几百kb的DNA片段。由于YAC非常不稳定，使用BAC变得越来越流行。我们尝试建立一种通过向受精卵中微量注射BAC来产生转基因小鼠的方法。我们发现，通过脉冲场凝胶电泳，去除含有BAC的琼脂糖凝胶部分（DNA约为100 ~ 200 kb），琼脂酶消化琼脂糖，可以分离BAC。微量注射BAC DNA的最佳浓度为1 ~ 1.5 ng/μl。虽然活产小鼠的数量比注射正常DNA的小鼠少，但转基因生产的总体效率是相同的，10 - 60%的活鼠转基因。由于大约165kb大小的BAC9似乎包含了完整的qkI基因，我们注射了这些BAC9并建立了两种转基因小鼠。这些小鼠与颤抖的小鼠交配，产生具有颤抖背景的转基因小鼠。这些转基因小鼠没有出现震颤表型。含有T基因的BAC也能挽救T表型。这些结果清楚地表明，BAC转基因可以成为检测用于转基因的BAC是否含有候选基因的有力工具。

Chromosome should be replicated without any loss of DNA at the end of chromosome and each one chromosome should be separated into daughter cells while cell division. To accomplish this process, at least three elements, replication origin, telomere, and centromere, are required. In yeast and E.coli, these elements are well characterized and are already cloned. Using these elements, it is now possible to construct artificial chromosome such as yeast artificial chromosome (YAC) or bacterial artificial chromosome (BAC). These artificial chromosomes can be used for isolating a large fragment of DNA spanning to a few hundreds of kb in BAC and a few magabase in YAC.As YAC is quite unstable, use of BAC is becoming more popular. We have tried to establish a method to produce transgenic mice by microinjecting BAC into fertilized eggs. We found that isolation of BAC can be done by pulse field gel electrophoresis, removal of agarose gel fraction containing BAC with about 100 to 200 kb DNA, and digestion of agarose with agarase. The optimum concentration of BAC DNA for microinjection was 1 to 1.5 ng/μl. Although the number of live born mice is lower than that in injection with normal DNA, the overall efficiency of transgenic production was the same and 10 to 60 % of live mice were transgenic. As BAC9 of about 165kb size seems to contain whole qkI gene, we injected these BAC9 and established two lines of transgenic mice. These mice were mated with quaking mice to produce transgenic mice with quaking background. These transgenic mice did not develop quaking phenotype. BAC containing T gene could also rescue the T phenotype. These results clearly suggest that BAC transgenesis can be a powerful tool to test whether the BAC used for transgenesis contains a candidate gene.