I tried preparing lentiviral particles with pLKO.1 system with psPAX2 packaging plasmid and pMD2.G envelope plasmid in ratio of 1: 0.75: 0.250. I tried twice with Lipofectamine LTX reagent in HEK293T cells. I didn't get the lentivirus shRNA, the shRNA construct of pLKO.1 system is effective as I have verified it with normal stable transfection and it did work.
Also I have used a LV-RFP positive control that also did not work. Can anyone please help me out?
Best guess would be that either one of the two packaging plasmids is messed up in some fashion or your transfections are not working. Given that PAX2 works for second generation viruses this should work (ie not an incompatibility issue). For the RFP vector did you see a lot of RFP expression in your transfected 293T cells? If not your problem is at the level of transfection. We use PEI transfections to make lentivirus because we make many viruses this is the most cost effective method. The procedure should be in here (DOI:10.1371/journal.pone.0076279).
turns out almost every ratio of these three plasmids gives us virus...they differ in the amount of virus though
Best guess would be that either one of the two packaging plasmids is messed up in some fashion or your transfections are not working. Given that PAX2 works for second generation viruses this should work (ie not an incompatibility issue). For the RFP vector did you see a lot of RFP expression in your transfected 293T cells? If not your problem is at the level of transfection. We use PEI transfections to make lentivirus because we make many viruses this is the most cost effective method. The procedure should be in here (DOI:10.1371/journal.pone.0076279).
turns out almost every ratio of these three plasmids gives us virus...they differ in the amount of virus though
Checklist:
1. Since the positive control is also not working - check all the packaging vectors at least with restriction digestions to see that you have the right stuff - if needed prepare all the plasmids new (starting from first glycerol stock or transform from the ORIGINAL (not from the one that did not work) pinch of DNA if possible)
2. Confirm that your transfection works (look for bright RFP/GFP-signal in packaging cells after transfection as suggested by David)
3. Healthy 293T cells can be efficiently transfected essentially with any method so you should also confirm that your packaging cells are not contaminated
If you can first exclude the above only then one should go into more detailed troubleshooting (eg. efficiency of the shRNA-construct, virus concentration, harvest times etc...)
Good luck!
It is absolutely mandatory that your 293T are free of mycoplasm, otherwise you won't get any virus. PEI transfection is perfect (PEI (Polysciences, Inc. Num. Cat: 24765) in NaCl 150 mM). Poly-lysin coating prior 293T seeding for transfection helps a lot. The day after transfection replace medium with DMEM 2% FCS (Complement inactivated) and you may booster virus production adding Sodium Butyrate at 5 mM final concentration at this step. Before, infection of the target cells we spin the supernatant and filter through a 40 micrometer cell strainer instead of using a syringe-attached filter device, that may retain lots of viral particles depending on filter membrane material used.
GOOD LUCK!!!
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