Construction of linear functional expression elements with DNA fragments created by site-specific DNA nickase, N.Bpu10 I, and exonuclease III.
A method to assemble linear expression elements for rapid gene expression is described. Primers containing target specific sequences and N.Bpu10 I nickase recognition sites were used to amplify promoter, open reading frame and terminator fragments. Amplified fragments were treated with N.Bpu10 I nickase and exonuclease III to generate overhangs for directional ligation. These fragments were ligated and further amplified with element-specific primers. The amplified DNA was transfected into mammalian cells for gene expression.
Xin
Wen
W
Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, PR China.
Zhang
Yi-Ming
YM
Xiao
Jin-Hua
JH
Huang
Da-Wei
DW
D000818:Animals; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053938:DNA Fragmentation / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D005240:Feasibility Studies; D005786:Gene Expression Regulation / Q000235:genetics*; D013006:Growth Hormone / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013728:Terminator Regions, Genetic; D014162:Transfection / Q000379:methods*
D017931:DNA Primers; D011994:Recombinant Proteins; D013006:Growth Hormone; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C061882:endodeoxyribonuclease Bpu10I; D003850:Deoxyribonuclease I; D015252:Deoxyribonucleases, Type II Site-Specific