Cationic lipid-mediated co-transfection of insect cells

Current co-transfection protocols use calcium phosphate precipitation for cultured insect (Sf-9) cells followed by multiple agarose overlays to purify recombinant baculoviruses (1). We report a more effective and convenient method that uses the cationic lipid N[1,1-(2,3-dioleyloxy)propoyl]-N,N,N,trimethylammonium chloride, or DOTMA (2, 3), for cotransfections of Sf-9 cells and uses 96-well microtiter plates to identify and purify recombinant baculoviruses. The cotransfection method is not pH sensitive and may be used with vector DNA from mini-lysate protocols. DOTMA is sold by Gibco/BRL under the trade name LipofectinTm. The cDNA genes for both the aand fl-chains of hemoglobin were cloned into the baculovirus transfer vector pAcYM1 (4) and verified by restriction enzyme digestion, partial DNA sequencing, and dot-blot hybridization (results not shown). One isg of CsCl-purified transfer vector for the a-chain of hemoglobin was mixed with 2Atg of AcMNPV DNA and 30 Ag of DOTMA in a polystyrene container. The solution was brought to a volume of 50 A1 with water, mixed, and allowed to sit at room temperature for 15 minutes. The DNA/DOTMA complex was mixed with 3 ml of serum-free Grace's medium supplemented to 0.33% lactalbumin hydrolysate and 0.33% yeastolate and added to a 25-cm2 flask containing 3 x 106 Sf-9 cells. The cells were then incubated without serum at 27°C for 3-24 hours. After this incubation, the medium was supplemented to 10% fetal bovine serum in a total volume of 6 ml and the cells incubated for a week at 27°C. 10-5 to 10-7 dilutions of the co-transfection medium were prepared and each dilution used to infect Sf-9 cells seeded in a 96-well microtiter plate at 1 x 104 cells/well. The plates were incubated at 27°C for a week. Each well was scored for the presence of viral infection and the viral titer of the cotransfection medium calculated by end-point dilution (1). We find that the viral titer from the co-transfection of Sf-9 cells using DOTMA is at least 20-fold greater than the titer obtained from the calcium phosphate precipitation method. The cells from each plate were lysed and screened with a 32P-labeled probe to identify recombinant viral samples. Figure la shows an autoradiograph of a nitrocellulose filter from a 96-well dot-blot hybridization of a plate of cells infected with a 10-5 dilution of the co-transfection medium. The percentage of recombinant virus present in the co-transfection medium is estimated to be 5-10%, a 5-50-fold increase in recombinant virus over the calcium phosphate precipitation method. We find that an overnight incubation of Sf-9 cells in the presence of DOTMA provides optimal recombinant virus production. The microtiter plate screening procedure was done a second time to purify recombinant virus from wild-type virus. This process was also performed for the fl-chain of hemoglobin. Purified recombinant viruses for both chains (Ha and Hfl) were used to infect 25-cm2 culture flasks of Sf-9 cells (1). Cells were collected at 72 hours post-infection, washed, resuspended in sample buffer, and analyzed on a 15% SDS-polyacrylamide gel. Figure lb shows that proteins identical in size to each chain are expressed in the infected cells.