Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse Brachyury locus
Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse Brachyury locus
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DOI：
10.1538/expanim.53.311
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发表时间：
2004-07-01
影响因子：
2.4
通讯作者：
Suzuki, M
中科院分区：
文献类型：
--
作者：
Abe, K;Hazama, M;Suzuki, M
使用YAC或BAC等大DNA的转基因扩大了功能基因组学的应用范围。在这里，我们描述了一种高效的BAC转基因方案，该方案采用了一种简单的BAC DNA制备方法，该方法采用了YAC DNA纯化方法。这种方法使我们能够从小规模培养的含有BAC的细胞中分离出足够数量和纯度的BAC DNA，用于显微注射。用这种方法制备的线性化BAC DNA已经产生了40多个创建者，其中85%的人含有完整的BAC转基因。相反，当注射环状BAC DNA时，观察到转基因整合率大约降低了三倍，获得的完整转基因整合较少。一组携带以这种方式产生的170kb BAC克隆的转基因小鼠成功挽救了小鼠brachyury(T)突变体的尾部和胚胎致死表型，进一步证明了该方法在小鼠基因组功能分析中的实用性。
Transgenesis using large DNA such as YAC or BAC has extended the range of applications in functional genomics. Here we describe an efficient BAC transgenesis protocol using a simple BAC DNA preparation method adopted from YAC DNA purification methods. This method allowed us to isolate BAC DNA from small scale culture of BAC-containing cells in sufficient quantity and purity for microinjection. More than 40 founders have been produced with linearized BAC DNA prepared by this method, and 85% of them contained intact BAC transgenes. In contrast, when circular BAC DNA was injected, an approximately three-fold reduction of transgene integration rate was observed and fewer intact transgene integrations were obtained. A line of transgenic mice carrying a 170-kb BAC clone generated in this way successfully rescued tail and embryonic lethality phenotypes of the mouse Brachyury (T) mutants, further demonstrating the utility of this method in functional analysis of the mouse genome.