I am currently trying to establish lentivirus production in HEK293T cell line. I have two packaging systems I could use:
- pLP1 / pLP2 / pVSV/G (from Invitrogen i think, we somehow have the plasmids separately)
- pCMVR8.92 (encodes gag/pol) / pRSV-rev (rev) / pMD.G (VSV env)
pCMVR8.92 is a gag-pol vector similar to pCMVR8.91 with a deletion in the Rev coding sequence afaik.
I have three different transfer vectors i would like to use:
- pLVX-dd-ZsGreen1 (Reporter System from Clontech)
- pLKO.1 (commonly used shRNA vector)
- pMF359-IRES-EGFP (constructed lentiviral vector based on pNL-EGFPU3, see this publication: http://nar.oxfordjou...e113#GNF112TB1)
I seeded 7*10^5 HEK293T cells on a 3.5cm dish (6-well plate) and on the next day i did transfection with MetafectenePRO (Biontex) with following DNA amounts:
1ug of all packaging plasmids and env-plasmid, 2ug of transfer vector (I only tried pLVX and pMF so far)
Transfection worked since i had green cells the next day (when I also changed the media). Efficiency was around 60% (of course i can only say this for the transfer vector).
Unfortunately the harvested supernatant could not infect HEK293T or human melanoma cells (I added 200 ul cleared but unconcentrated supernatant to 80% confluent cells in a 6 well plate or 1.2 ml sup to 100mm plate)
Does anyone see an obvious error? Why do most protocols use Calcium-phosphate method for the transfection? I have a protocol from my lab which uses FuGene, but I think this is basically the same as MetafectenePRO.
What about the plasmid ratios? What i have seen online (e.g. lentiweb.com) people apply much transfer vector, less packaging plasmids, and even less envelope plasmid. Why? wouldn't it make more sense to make sure that every cell that takes up the transfer vector really contains the packaging and env plasmids? (e.g. make a ratio of 1:5:5:5 of transfer-vector:packaging-plasmids)
Any further suggestions?
Thanks for any answer.
Edited by loucash, 02 June 2010 - 01:11 AM.