[Construction of a dual-luciferase co-expression vector and its characteristics in vitro and in vivo].
A novel dual luciferase expression vector was designed and its expression characteristics were studied in vitro and in vivo. Firstly, the Gluc and Fluc genes were connected via the TaV 2A sequence by overlaping PCR, and inserted into the expression vector pAAV2neoCAG, obtaining the recombinant plasmid pAAV2neoCAG-Gluc-2A-Fluc. Then pAAV2neoCAG-Gluc-2A-Fluc was transfected into BHK21 cells and the activity of Gluc and Fluc in the supernatant and cell lysates were assayed respectively. Results showed that both Gluc and Fluc were expressed successfully. The Gluc was mainly detected in the culture media while the Fluc was mostly within cells. The activity of Gluc in the supernatant increased gradually with time while the Fluc activity in cells almost kept stable. To investigate the expression of pAAV2neoCAG-Gluc-2A-Fluc in vivo, the plasmid was hydrodynamically injected into BALB/c mice through tail vein. The Gluc activity was assayed in a small volume of blood taken by tail vein at different time points. Results showed that Gluc was expressed stably at least 7 days. Live bioluminescence imaging technology was used to compare the expression characteristics of Gluc and Fluc. Whole body imaging was seen when coelenterazine, a specific substrate for Gluc, was injected, and the imaging signals decreased rapidly within 10 minutes. Liver imaging was showed when Flue specific substrate named D-Luciferin was injected, and the imaging remained stable at least for half an hour. The dual luciferase expression vector pAAV2neoCAG-Gluc-2A-Fluc combines the advantages of the secreted report gene Gluc and the non-secreted report gene Fluc, and will provide a new tool for cell labeling and tracing.
Fu
Xin-Yang
XY
Department of Urology, Nan fang Hospital, Southern Medical University, Guangzhou 510515, China.
Wang
Gang
G
Tian
Wen-Hong
WH
Chen
Su-Yun
SY
Dong
Xiao-Yan
XY
Wu
Xiao-Bing
XB
Tan
Wan-Long
WL
D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D003445:Crustacea / Q000201:enzymology; D048888:Fireflies / Q000201:enzymology; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data
D008156:Luciferases