Assembly of linear functional expression elements with DNA fragments digested with asymmetric restriction endonucleases.
A three-step method is described to assemble linear functional expression elements for rapid gene expression. In step one, a eukaryotic promoter, an open reading frame and a transcription terminator are PCR-amplified individually with primers containing recognition sites of asymmetric restriction endonucleases. In step two, the three DNA fragments, digested with corresponding restriction endonucleases, are directionally joined by T4 DNA ligase to assemble linear functional expression elements. In step three, the resultant linear functional expression elements are amplified with element-specific primers by a second round PCR, followed by transfection into Chinese hamster ovary cells for gene expression.
Xin
Wen
W
Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, P.R. China.
Ma
Jing
J
Huang
Da-Wei
DW
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D011088:DNA Ligases / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry*; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D005240:Feasibility Studies; D005786:Gene Expression Regulation; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D013728:Terminator Regions, Genetic; D014162:Transfection / Q000379:methods*
D017931:DNA Primers; D004262:DNA Restriction Enzymes; D011088:DNA Ligases