A. Preparation (previous day)
Seed 2 - 3 × 106 of 293 cells or 293T cells into one 6 cm φ gelatin or collagen coated dish, and prepare recombinant retrovirus vector plasmid.
Purify the vector plasmid to a degree sufficient for transfection. Use CsCl gradients equilibrium ultracentrifugation method or an appropriate DNA preparation kit. B. Transfection (1st day)
Bring the transfection reagents and sterilized distilled water to room temperature. Operate the following steps avoiding any contamination.
1. Prepare the following vector mixture in a sterilized 5.0 ml round-bottomed tube (polystylene).
2. Change the culture medium to DMEM with 10% FBS and 25μM chloroquine (3 ml).
3. Form calcium phosphate precipitation and transfect to cells.
1) Suck up 500 μl of Transfection Buffer using electoric pipetter.
2) Add the buffer gently to the vector mixture with shaking the tube.
3) Bubble the solution immediately by using exhaust of pipetter for 10 - 20 sec. to accelerate the formation of calcium phosphate precipitation.
4) Within 1 - 2 min., drop the solution onto the cell dish evenly and rock the dish gently several times.
5) Incubate for 7 - 11 hours in 5% CO2 incubator at 37°C.
When calcium phosphate precipitation is formed successfully,it looks like powder-snow on the surface of cells under a microscope.
6) Remove 3 ml of the medium from the dish, and then add 4 ml of fresh DMEM with 10% FBS. (Complete removal of culture medium may reduce the transfection efficiency, so it is recommended to leave 1 ml of medium in the dish. C. Exchange of medium (2nd day)
After 24 hours from transfection, change the culture medium again to DMEM with 10% FBS (4 ml).
D. Virus solution (3rd day)
After 48 hours from transfection, collect the supernatant of culture medium and filtrate with 0.45 μm sterilized filter. For storage, the virus solution must be dispensed each in a small amount and stored at - 80°C. Avoid repeating freeze-thaw cycles.
Note : The titer of the recombinant virus prepared using this kit is varied by size of inserted target gene in retrovirus vector, efficiency of transfection,etc. Generally, the titer is transiently 105 - 107 infectious units/ml. 293T cells, which were made by introducing SV40 T antigen gene into 293 cells, are useful for transient transfection and can produce higher titer virus than 293 cells.