Hello,
I am trying to generate a PC-3 Prostate Cancer Cell line expressing PSMA and Luciferase. I modified the pBOBI Fluc vector (https://www.addgene.org/170674/) and cloned the PSMA gene and an IRES Luciferase into the MCS.
I have been using a 3rd generation packaging system with pMDL, pREV, and pVSV-G (Addgene #12251, #12253, #8454). I tried a 1:1:1:1 ration for my transfection in 293T and I've done the protocol both with Lipofectamine 3000 and PEI, but I haven't been able to infect my PC-3 cells as they all die upon antibiotic selection. This plasmid has a BleoR gene, so I've been using Phleomycin as the selection antibiotic.
I really don't know what could be wrong with it. If someone has any insights I would be very grateful.
I suggest you use the following protocol from Antonia Follenzi and Luigi Naldini. I tried it and it worked:
Chapter HIV-based vectors
Use a 1:3 ratio with PEI for lentiviral production. Avoid repeated freeze-thaw cycles after storing the lentiviral stock at -80°C. If you're using Polybrene for transduction of PC-3 cells, make sure to use the lowest effective concentration, as these cells can be sensitive to it.
Dear Paula Iglesias Matesanz
There could be several causes to the lack of viral transduction.
• the plasmid stoechiometry that is not optimized (1:1:1 may not be adequate)
• the plasmid design may have an issue, meaning that one of the sequence is not perfect (cloning may have failed)
• the transfection reagent and this is the object of my answer.
The last point is crucial to us since we are specialized in transfection reagents and we have developed HYVIR for lentiviral virus production.
■ Not all the transfection molecules are able to induce correct viral production : either in terms of completion of viral particles, ratio of active/non active particles, or in forming efficient transfection complexes.
this results in a dramatically low final MOI and following experiments are thus non reliable.
→ In order to clear the last point I would like to offer you to try our HYVIR transfection reagent, to be sure that the problem does not come from the transfection of the plasmids.
If you need any information or if you want to try HYVIR, please do not hesitate to contact me directly at:
I will be glad to send you a sample,
best regards,
Cedric
Hi Samaneh Ghadami , I've been checking the plasmid and realized that the BleoR gene is outside of the LTRs, meaning that the viruses won't harbor the resistance gene. You'll need to insert the resistance gene into the LTR-delimited sequence to get resistant clones through infection.
Hi Daniel Martín Thank you for answering! This might be the problem. So the only part of the plasmid that will be packed is the one delimited by the LTRs, right?
Thank you for your insights Samaneh Ghadami . I'll look up the protocol you suggested.
That's it, Paula Iglesias Matesanz only those elements wrapped between the LTRs will be packaged into the lentiviral particles. Whatever is outside can be used in transfection or bacterial transformation but not in transduction.
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