Xiao Jin-Hua, Xin Wen, Liu Yong-Jie, Murphy Robert W, Huang Da-Wei
Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
Mol Biotechnol. 2007 Jan;35(1):15-22. doi: 10.1385/mb:35:1:15.
Linear expression constructs can facilitate gene function studies. We describe a method to generate linear expression constructs for mammalian cells by one-step polymerase chain reaction (PCR) with vaccinia DNA topoisomerase I (TOPO). Cytomegalovirus (CMV) 5' promoter, the gene of interest, and V5 bovine growth hormone (BGH) polyA 3' terminator elements were PCR-amplified with target-specific primers containing vaccinia DNA TOPO-specific sequence and complementary sequence to each other. We amplified specific and complementary sequences. These three elements were directionally joined with vaccinia TOPO. The joined products were then directly transfected into Chinese hamster ovary cells. Compared with the transfection of supercoiled plasmids, comparable expression signals were obtained for green fluorescent protein, chloramphenicol acetyltransferase, and beta-galactosidase proteins using Western blots. This is a quick and efficient method to generate linear expression constructs. Unlike Invitrogen TOPO Tools, our method avoided the secondary round of PCR and more rapidly yielded correct joining products. This method can be easily used in the function test of uncharacterized open reading frames.
线性表达构建体有助于基因功能研究。我们描述了一种通过用痘苗DNA拓扑异构酶I（TOPO）进行一步聚合酶链反应（PCR）来生成用于哺乳动物细胞的线性表达构建体的方法。用含有痘苗DNA TOPO特异性序列且彼此互补的靶标特异性引物对巨细胞病毒（CMV）5'启动子、目的基因和V5牛生长激素（BGH）聚腺苷酸3'终止子元件进行PCR扩增。我们扩增了特异性和互补序列。这三个元件与痘苗TOPO定向连接。然后将连接产物直接转染到中国仓鼠卵巢细胞中。与超螺旋质粒转染相比，使用蛋白质免疫印迹法对绿色荧光蛋白、氯霉素乙酰转移酶和β-半乳糖苷酶蛋白获得了相当的表达信号。这是一种快速有效的生成线性表达构建体的方法。与Invitrogen TOPO Tools不同，我们的方法避免了第二轮PCR，并且更快地产生了正确的连接产物。该方法可轻松用于未表征开放阅读框的功能测试。
