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	Generation of linear expression constructs by one-step pcr with vaccinia dna topoisomerase i
	Xiao, Jin-Hua; Xin, Wen; Liu, Yong-Jie; Murphy, Robert W.; Huang, Da-Wei
刊名	Molecular biotechnology
	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
出版者	HUMANA PRESS INC
WOS记录号	WOS:000244189700002
内容类型	期刊论文
URI标识	http://www.corc.org.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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