Generation of linear expression constructs by one-step PCR with vaccinia DNA topoisomerase I.
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.
Xiao
Jin-Hua
JH
Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
Xin
Wen
W
Liu
Yong-Jie
YJ
Murphy
Robert W
RW
Huang
Da-Wei
DW
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter; D005814:Genes, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004264:DNA Topoisomerases, Type I