IDENTIFICATION OF DISEASE GENE USING BAC TRANSGENIC MICE

染色体的复制应在染色体末端不丢失DNA，每条染色体在细胞分裂时应分离成子细胞。要完成这一过程，至少需要三个要素，即复制起点、端粒和着丝粒。在酵母和大肠杆菌中，这些元件都有很好的特性，并且已经被克隆。利用这些元件，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)。这些人工染色体可以用来分离BAC中跨度达几百kb的大片段DNA和YAC中的少量大碱基。由于YAC很不稳定，BAC的使用越来越普遍。我们试图建立一种将BAC显微注射到受精卵中来产生转基因小鼠的方法。我们发现，BAC的分离可以通过脉冲场凝胶电泳法，去除含有约100~200kb DNA的BAC的琼脂糖凝胶部分，然后用琼脂糖酶消化。结果表明，注射BACDNA的最适浓度为1~1.5 ng/μL，虽然活仔数低于注射正常DNA的小鼠，但转基因产品的总体效率相同，转基因小鼠的成活率为10%~60%。由于大小约165kb的BAC9似乎含有完整的qkI基因，我们将这些BAC9注射进去，建立了两个转基因小鼠系。这些小鼠与颤抖的小鼠交配，产生背景颤抖的转基因小鼠。这些转基因小鼠没有出现颤抖表型。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是否包含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.