1ME A17R11 WI238 DMS2114 JC L2929 P815 PT67

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1 Chinese Journal of Biotechnology Vol. 20 No. 1 January (, ), BacV2CMV2EGFPA,Sf9, 20, 12,7,1 CMV,,,LipofectAMINE CMV EGFP pcdna3112egfp, CMV EGFP, CMV CMV,,,,,, Bac2to2Bac 2, CMV,,, Q813 A (2004) , [1-3 ] Hofmann, [4 ] Boyce [5 ],, [6,7], CMV, ( GFP),, GFP, CMV, CMV (egfp) BacV2CMV2EGFPA, [8 ], CMV : Sf9 Invitro2 gen CV B Hep G2 PLCΠPRFΠ5 BNL 1ME A17R11 WI238 DMS2114 JC L2929 P815 PT67 : , : : 863 (No. 2001AA628120) 3 Tel : ; Fax : ; E2mail : xmu. edu. cn

2 74 20 ATCC, Hela CHO NIH3T3 Raji CNE MCF27 BGC2223 pcd2 NA3112EGFP LCL2cm BacV2 CMV2EGFPA : Grace DMEM RPMI1640 MEM Invitrogen OXOID [9 ] : Grace 10 % 56, 50mL Sf9, 80 %, Sf9, 1 3 3,27 3d - 80 Sf9,4d, 1 20, :Sf9 80 % 6, 28 1d, 10 Sf9, 1h 2mL Grace (018gΠmL), 28 5d, 4, ( Plaque forming unit,pfu, ) : 24, h, 500 L Sf9, 37 8h 48h LipofectAMINE : 70 % 24, 37 12h 1 g 100 L ( solution A), LipofectAMINE 2 L 100 L (solution B), solution A B, 45min 24, 1mL 3, 500 L, solution A B,37 6h 24, 500 L 48h : Nikon TE200,Nikon coolpix :,1 500rΠmin 5min, 5 % PBS 200 (Beckman Coulter EPICS XL),488nm,525nm, EX2 PO ,, egfp (B ), B 2 % B B ; B Hep G2 CV1 P815 PLCΠPRFΠ 5 143B CHO Hela Raji JC NIH3T , 12,7,1 BacV2CMV2EGFPA Sf9 ( PFUΠmL) 1 (moi = 50),12h,48h, 1,Sf9, 212 CMV, EGFP,, CMV, CMV, CMV,,

3 1 : 75 CMV EGFP, CMV pcdna3112egfp CMV EGFP LipofectAMINE pcdna3112egfp CHO2K1 JC PT67 293, 48h egfp, 2, CMV EGFP 1 BacV2CMV2EGFPA Table 1 Comparison of mammalian gene2transfer and expression efficiencies of Baculovirus BacV2CMV2EGFPA Organism Tissue Growth properties Transduction efficiency Mean fluorescence intensity Human WI238 Lung fibroblast Adherent % Hela Adenocarcinoma Adherent % 4817 Hep G2 Hepatocellular carcinoma Adherent % Kidney Adherent % 4614 PLCΠPRFΠ5 Hepatoma Adherent % B Osteosarcoma Adherent % 5418 MCF27 Gastric carcinoma Adherent % 7213 BGC2223 Breast carcinoma Adherent % 6614 DMS 114 Small cell lung cancer Adherent % 9218 CNE Nasopharyngeal carconoma Suspension 0150 % 718 Raji Burkitt s lymphoma Suspension 1139 % 414 LCL2cm B lymphocyte Suspension 0169 % 914 Mouse BNL 1ME A17R11 Liver Adherent % 3913 CHO2K1 Ovary Adherent % 2612 L2929 Subcutaneous connective tissue Adherent % 3512 JC Adenocarcinoma Adherent % 2416 PT67 Embryo fibroblast Adherent 7141 % 1214 NIH3T3 Embryo fibroblast Adherent 7142 % 1316 P815 Mastocytoma Suspension 0168 % 516 Monkey CV1 Normal kidney Adherent % pcdna3112egfp Table 2 Comparison of mammalian gene2transfer and expression efficiencies of pcdna3112egfp transfected cell lines Organism Tissue Growth properties Transduction efficiency Mean fluorescence intensity Human WI238 Lung fibroblast Adherent % 5813 Hep G2 Hepatocellular carcinoma Adherent % 7914 Hela Adenocarcinoma Adherent % Kidney Adherent % 8616 Raji Burkitt s lymphoma Suspension 5156 % 1416 LCL2cm B lymphocyte Suspension 7162 % 1714 Mouse BNL 1ME A17R11 Liver Adherent % 1911 CHO2K1 Ovary Adherent % 4816 JC Adenocarcinoma Adherent % 2218 PT67 Embryo fibroblast Adherent % 3116 NIH3T3 Embryo fibroblast Adherent % 3513 P815 Mastocytoma Suspension % , 3, ( t = , P <

4 ),,,,, ( t = , P < 0105),, 3 Table 3 Comparison of the transduction efficiencies in adherent and suspend culture cells of different organisms transduced with BacV2CMV2EGFPA Adherent Suspend Primate Total 10 3 Mean transduction efficiency % % 1112 % 0153 % Mice Total 6 1 Mean transduction efficiency % % 0161 % 214 1,,, Hep G2 PLCΠPRFΠ5 ( t = , P < 0105), WI238 (SV40 ) DMS 114 ( t = , P < 0105) 3,, Sf9,,, 20, Sf9,,, 4,3,, 2 %, Condreay [10 ],,, CMV EGFP,CMV EGFP, CMV, Hela WI238, 1Π5, DMS 114 Hela,

5 1 : 77,,,, CMV,,,,,,, REFERENCES( ) [ 1 ] Merrihew RV, Clay WC, Condreay JP et al. Chromosomal integra2 tion of transduced recombinant baculovirus DNA in mammalian cells. J Virol, 2001, 75 : [ 2 ] Kost TA, Condreay JP. Recombinant baculoviruses as mammalian cell gene2delivery vectors. Trends Biotechnol, 2002, 20(4) : [ 3 ] Huser A, Hofmann C. Baculovirus vectors : novel Mammalian cell gene2delivery vehicles and their applications. Am J Pharmacogenom2 ics, 2003, 3 :53-63 [ 4 ] Hofmann C, Sandig V, Strauss M et al. Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc Natl Acad Sci USA, 1995, 92 : [ 5 ] Boyce FM,Bucher NLR. Baculovirus mediated gene transfer into mammalian cells. Proc Natl Acad Sci USA, 1996, 93 : [ 6 ] Shoji I, Aizaki H, Tani H et al. Efficient gene transfer into various mammalian cells, including non2hepatic cells, by baculovirus vec2 tors. J Gen Virol, 1997, 78 : [ 7 ] Yap CC, Ishii K, Aoki Y et al. A hybrid2baculovirus2t7 RNA poly2 merase system for recovery of an infectious virus from cdna. Virolo2 gy, 1997, 231 : [ 8 ] Cheng T( ), Xu CY( ), Wang YB ( ) et al. Rapid and efficient expression of foreign genes in mammalian cells by baculovirus vectors. Chinese Journal of Biotechnology ( ), 2003, 19(5) : [ 9 ] Bac2to2Bac baculovirus expression systems instruction manual. In2 vitrogen life technologies, 2002 [10] Condreay JP, Witherspoon SM, Clay WC et al. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proc Natl Acad Sci USA, 1999, 96(1) : Mammalian Gene2transfer and Expression Efficiencies of Baculovirus Bac V2CMV2EGFPA XU Chen2Yu CHENG Tong LU Wu2Xun CHEN Min WU Ting WANG Ying2Bin ZHANGJun 3 XIA Ning2Shao ( Key Laboratory of Cell Biology and Tumor Cell Engineering of Ministry of Education, Xiamen University, Xiamen , China) Abstract It has been reported that baculoviruses could serve as a new gene2transfer vehicle for mammalian cells. We have pre2 viously constructed recombinant baculovirus BacV2CMV2EGFPA and have proven that mammalian cells could be effectively in2 fected by the recombinant baculovirus. In this report, we studied the efficiency of baculovirus to deliver exogenous gene into twenty mammalian cells, including twelve human cell lines ( WI238, Hela, HepG2, 293, PLCΠPRFΠ5, 143B, MCF27, BGC2 223, DMS 114, CNE, Raji, LCL2cm), seven murine cell lines (BNL 1ME A. 7R. 1, CHO2K1, L2929, JC, PT67, NIH3T3, P815) and one monkey cell line ( CV1). Results showed that most mammalian cell lines could be transduced by the recombinant baculovirus, the transduction efficiencies of the human and monkey cell lines were markedly higher than that of murine cell lines, and the transduction efficiencies in adherent culture cell lines higher than that of suspend culture cell lines, implying that the in2 fection efficiency of the baculovirus may be correlative with the organism used and the growth properties of the cell lines. The plasmid pcdna3. 12EGFP, which contains the CMV promoter and EGFP reporter gene, was next transfected by LipofectAMINE into a number of mammalian cells, especially those cells that were low in the baculovirus transfection. Results showed that the CMV promoter could effectively direct the expression of the reporter gene in these mammalian cells. Therefore the gene2expres2 sion efficiencies in different mammalian cell lines by the recombinant baculovirus which contains the same CMV promoter were dictated by the ability of the baculovirus to enter the cell lines. This study suggested that the recombinant baculovirus vector is more suitable for gene expression in primate adherent culture cells than in murine cells and suspend culture cells. Key words Bac2to2Bac baculovirusπinsect cell expression system,cmv promoter,green fluorescent protein, FCM,mammalian cells Received : This work was supported by Grant from 863 Program (No. 2001AA628120). 3 Corresponding author. Tel : ;Fax : ; E2mail : xmu. edu. cn