pCMV-Gag-Pol载体

双质粒系统逆病毒包装质粒pCMV-Gag-Pol使用方法——逆转录病毒包装与转染方法


Day 1

1.      Plate 5 x 105 293T cells in 6 cm2 dishes containing 5 mL of media. (This can be scaled up if 

desired)


Day 2

1.      Set up (use polypropylene tubes for this; polystyrene 

tubes DO NOT work!):

           1 µg retroviral DNA encoding gene X
           1 µg packaging plasmid Mix
                the packaging plasmid (pCMV-Gag-Pol) at a 8:1 ratio with the envelope plasmid (pCVM-VSV-

G)-a total of 1µg
           DME without serum to 94μL total
           6 μL Fugene

       Mix and wait 15 to 30 minutes at room temperature

       Add to 293T cells without touching the sides of the dish (DO NOT CHANGE MEDIA)

       If you are using amphotropic virus then move immediately to BL2+ in a secondary container, 

which has an absorbent material. 

       (This does not mean a couple of hours; it means Immediately!).  The rest of this protocol is 

the same for all viruses---the BL2+ safety practices are in place if you are using amphotropic 

viruses 


Day 3

1.      Change the media to whatever media you wish to use when infecting target cells. 293T cells 

are easily detached so remember not to put the media directly onto to cells, but rather 

“run” it down the side of the dish. Remember that you will get the highest titer virus 

when your cells are “happy.”

2.      Plate out your target cells


Day 4

1.      Remove the medium from the 293T cells and use a 0.45 u syringe filter to remove any 293T 

cells. DO NOT use the 0.2 u filter, as it is likely to shear the envelope from your virus
making it noninfectious

        Note: After filtering, the filter should be removed and placed in the biohazard bag in the 

hood and the syringe rinsed with bleach and decontaminated for a minimum of 20 min. It is useful to 

place a plastic beaker with bleach in the hood in advance

2.      Add 8 to 10 μg/ml of polybrene (Hexadimethrine bromide) or protamine sulfate to the target 

cells

3.      Carry out infection for 1 to 4 hours. Remove virus and replace with fresh media

        Note: If you wish to do a second infection the following day, it is important to put fresh 

media on the cells and not let the virus remain on the cells overnight. The media contains huge 

amounts of envelope, both associated and unassociated with viral particles, which will bind all the 

cell surface receptors required for virus adsorption, resulting in their down-regulation. Hence, if 

you don’t change the media after the initial infection, very few receptors will be available 

for the next round of infection. In addition, very few cells tolerate the presence of high levels of 

the VSV-G envelope for extended periods of time (i.e. a lot of your cells may die)


Day 5 +

1.      Allow the cells to recover and begin to express the virus-encoded genes. The cells usually 

require 48 hours for this to occur

2.      Add drug if you are scoring for the presence of a vector that carries a drug resistance 

marker. Prior to this step it is advisable that you titrate the drug to be used for selection in 

order to know precisely how much to add. In addition, it is necessary to bring an extra plate of 

uninfected cells (often referred to as “canaries”) which will function as a positive 

control in the kill assay.  Add drug to both plates. When your canaries are dead, you can remove the 

drug.