BD Biosciences Clonetech’s Amphopack HEK 293 Retroviral Packaging System is an easy, safe and effective method to package your gene of interest to produce high titer, recombinant retroviruses. Bleomycin and puromycin resistance was used to separately introduce viral gag-pol and env genes into the HEK cells required for packaging, processing, reverse transcription and intergration of recombinant genes. Moreover, neomycin and hygromycin selection can be used to produce stably transfected packaging cells with a high titer yield.
The Amphopack 293 cells were derived from monkey embryonic kidney cells and are easily transfected with many different transfection reagents. These cells express the 4070A amphotropic envelope protein and the packaged virus will recognize the Pit2 receptor found on many mammalian cells. Retroviral expression vectors that have the psi- packaging signal can easily be used to produce infectious viral particles.
In our lab, we use this system to easily produce modified cell lines. The HEK 293 cells are easy to culture in high glucose DMEM supplemented with 10% fetal bovine serum. We trypsinize the cells and seed them at 106 cells per 6cm dish. The following day, we change the medium in the morning and then transfect four hours later with up to 10ug of our retroviral expression vectors. Two hours later, we add additional media and incubate overnight. The following morning we change the media and then allow the cells to incubate for up to 72 hours. We collect the medium to titer it before we use it. Infecting target cells with packaged retrovirus is very easy and requires one extra component called polybrene to help with virus and cellular association.
In our hands, we usually generate 107 cfu although the company guarantees that you will produce 106 cfu as your viral titer. Our high titer may be due to that fact that we use 10ug of DNA for transfection and that we transfect for at least 84 hours after the addition of DNA. Before you titer the virus, it is imperative that you set up an antibiotic killing curve so you know what concentration of antibiotic to use in order to properly titer your virus. This assay takes approximately 10 days to perform and uses NIH3T3 cells.
The major complaint that I have with this packaging system is that you have to transfect many 6cm dishes in order to create a substantial stock of retrovirus. I usually set up 25 6cm dishes to transfect for each viral DNA I wish to package. Other, that that one downfall, I believe that BD Biosciences’ Amphopack Retroviral Packaging System is a very easy and user friendly method to produce high titers of retrovirus used in gene transfer.
Sharon Yavrom
Graduate Student
University of Southern California