Hi everyone,
I've been recently trying to produce ecotropic retroviral vectors using the Platinum-E (Plat-E) cell line thorugh calcium phosphate percipitation method. Although the transfection seems to be working very well (as visualized by the presence of GFP under fluorescent microscope), the transduction efficiency has been almost zero when trying to titrate the virus-containing supernatant on NIH3T3 cells.
I have suspected that my Plat-E cell line is not functioning properly. As I have noticed in the catalogue for Plat-E from Cell Biolabs, as well as in the original article which has first reported the development of the Plat-E cell line, "these cells can produce retroviruses in the presence of drug selection for at least 4 months". Since the genes encoding for gag, pol and env are inserted along with puromycin and blasticidin resistance genes, I have suspected that the absence of these two antibiotics may have led to loss of genes encoding these structural proteins and hence may account for the inability of the cells to produce functional viral particles.
The supplier from which I purchased the Plat-E cell line did not mention that the use of puromycin and blasticidin is compulsory. I just want to ask from anyone who had experience working with these cells, are these antibiotics necessary in their culture medium? Have you got positive results in the absence of these two antibiotics?
Hi Mina, I'm currently battling the same/similar issue here, unfortunately I don't have an answer yet but I was wondering if maybe you found a solution in the meantime?
In the past, I have done it successfully without antibiotic selection. However, I haven't done it in a while and now have another batch of cells that doesn't work. So I will give it a try with the antibiotic selection and hope it works!
Hi Sarina,
After checking many protocols and repeating the transduction procedure over and over, I came to the conclusion that Plat-E cells cannot function without antibiotic pressure, or at least they must be passaged a few times in the presence of antibiotics from time to time.
I did not have puromycin and blasticidin and they both were too expensive for me to buy (more expensive than the cell line itself!), so I started over with HEK293T and the helper plasmid pCL-Eco along with my plasmid, and it worked.
Good luck :)
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