中国科学院机构知识库网格
Chinese Academy of Sciences Institutional Repositories Grid
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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