Generation of linear expression constructs by one-step pcr with vaccinia dna topoisomerase i
文献类型：期刊论文
| 作者 | Xiao, Jin-Hua; Xin, Wen; Liu, Yong-Jie; Murphy, Robert W.; Huang, Da-Wei |
| 刊名 | Molecular biotechnology
 |
| 出版日期 | 2007 |
| 卷号 | 35期号:1页码:15-22 |
| 关键词 | Topoisomerase i Linear expression constructs (lec) Gene expression One-step pcr |
| ISSN号 | 1073-6085 |
| 通讯作者 | Huang, da-wei(huangdw@ioz.ac.cn) |
| 英文摘要 | 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. there 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. |
| WOS关键词 | ELEMENTS ; BINDING ; CLONING |
| WOS研究方向 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| WOS类目 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 语种 | 英语 |
| WOS记录号 | WOS:000244189700002 |
| 出版者 | HUMANA PRESS INC |
| URI标识 | http://www.irgrid.ac.cn/handle/1471x/2381995 |
| 专题 | 中国科学院大学 |
| 通讯作者 | Huang, Da-Wei |
| 作者单位 | 1.Chinese Acad Sci, Inst Zool, Beijing 100080, Peoples R China 2.Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China 3.Shandong Agr Univ, Plant Protect Coll, Shandong 271080, Peoples R China 4.Royal Ontario Museum, Dept Nat Hist, Toronto, ON M5S 2C6, Canada |
| 推荐引用方式 GB/T 7714 | Xiao, Jin-Hua,Xin, Wen,Liu, Yong-Jie,et al. Generation of linear expression constructs by one-step pcr with vaccinia dna topoisomerase i[J]. Molecular biotechnology,2007,35(1):15-22. |
| APA | Xiao, Jin-Hua,Xin, Wen,Liu, Yong-Jie,Murphy, Robert W.,&Huang, Da-Wei.(2007).Generation of linear expression constructs by one-step pcr with vaccinia dna topoisomerase i.Molecular biotechnology,35(1),15-22. |
| MLA | Xiao, Jin-Hua,et al."Generation of linear expression constructs by one-step pcr with vaccinia dna topoisomerase i".Molecular biotechnology 35.1(2007):15-22. |
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来源：中国科学院大学
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