[Rapid and efficient expression of foreign genes in mammalian cells by baculovirus vectors].
The baculovirus insect cell expression system has been used extensively for the expression of recombinant proteins in insect cells. Recently, reports have described that recombinant baculoviruses can transduce a broad spectrum of primary and established mammalian cells, which shows the baculoviruses could serve as a new gene-transfer vehicle for mammalian cells. In this report, we further research the modification of baculovirus vector and the way to deliver exogenous gene into mammalian cells. On the base of Bac-to-Bac baculovirus insect cell expression system, two recombinant baculoviruses (BacV-CMV-EGFPA, BacV-CMV-EGFPB) were constructed containing different direction of CMV promoters which controll the expression of a reporter gene (EGFP). We found that CMV promoter could direct expression of reporter gene in Sf9 cells with relatively low efficiency. The culture supernatant of Sf9 cells which have been infected by the recombinant baculoviruses for four days were collected and the titers of the viruses in culture supernatant were determined by plaque assay on Sf9 cells. The HepG2 cells, an human hepatocellular carcinoma cell line, were directly incubated with the collected culture supernatant which contains the recombinant baculoviruses for 8 hours in 37 degrees C CO2 incubator (moi = 100). Twenty-four hours post transduction the efficiencies of gene-transfer and expression were analyzed by flow cytometry (FCM) which detect the green fluorescence of individual cells. Results show that these two recombinant baculoviruses have similar gene-transfer and expression efficiency in HepG2 cells, which means the direction of CMV promoters has no effects on reporter gene expression. The optimal transduction conditions of incubating the mammalian cells with the culture supernatant of Sf9 cells infected by recombinant baculoviruses for four days were determined by FCM assay in HepG2 cells. The HepG2 cells inoculated in 24-well plate (5 x 10(4)/well) were incubated with the culture supernatant (BacV-CMV-EGFPA, 1.2 x 10(7) pfu/mL) serially diluted by DMEM culture medium containing 10% FBS and the transduction times ranged from 1 to 24 hours. Twenty-four hours post transduction the efficiencies of gene-transfer and expression were analyzed by FCM. Results show that incubating the target cells with the 1:1 diluted culture supernatant (moi = 50) for 12 hours in 37 degrees C CO2 incubator would achieve the highest infection and expression efficiency with the least impairment on cell viability. We compared the gene-transfer and expression efficiency of recombinant baculovirus in HepG2 and CV1 cells with lipofectAMINE and recombinant retrovirus system, results show that under the similar conditions the recombinant baculovirus could achieve the highest gene-transfer and expression efficiency than the other two systems. So we can draw a conclusion that directly incubating the mammalian cells with the culture supernantant of the infected Sf9 cells could serve as a very convenient way for rapid and efficient expression of foreign gene in mammalian cells.
Cheng
Tong
T
Key Laboratory of Cell Biology and Tumor Cell Engineering of Ministry of Education, Xiamen University, Xiamen 361005, China.
Xu
Chen-Yu
CY
Wang
Ying-Bin
YB
Chen
Min
M
Wu
Ting
T
Xie
Xiao-Yan
XY
Zhang
Jun
J
Xia
Ning-Shao
NS
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D000882:Haplorhini; D056945:Hep G2 Cells; D006801:Humans; D008957:Models, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics; D018411:Spodoptera
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins