Can anyone suggest methods for efficient transduction of activated primary human T cells with pWPI bicistronic vector based 2nd gen lentivirus carrying a chimeric receptor and GFP? 293T cells give 85-90% GFP and transgene expression by 48hr by flow but no significant transduction of activated T cells in presence of polybrene and spinoculation followed by overnight incubation.
Hi Junaid,
I have had quite a lot of experience with generating lentivirus to transduce primary human T cells. Its actually a lot difficult than people suggest. It took me a whole year of optimisation to achieve good transduction (70-85% routinely). I don't know if you have seen my paper attached but it is the method that I use routinely. Are you able to give me more information on exactly the protocol you use? Do you concentrate your virus? What MOI do you use? and war cells did you use to titrate your virus?
Article Simplified production and concentration of lentiviral vector...
I have used 2 different 2nd generation packaging systems for lentivirus production - one of them usually works better than the other for any given cell line. Here are the two that I've used with amount that I transfect into a 10cm plate of 293Ts:
1) Transfer vector - 10ug
pMD2.G-5ug
psPAX2-5ug
2) Transfer vector - 8ug
pCMVΔ8.91 - 6ug
pCMV-VSVG - 4ug
Hope this helps!
Thanks Adam and Ramon for your suggestions!
Adam — I have mailed you a detailed protocol that I use for making the virus.
Ramon — I have used 2 different systems with 2nd gen. I have tried pLVX-EF1a-ZsGreen1 from Clontech with their packaging mix for 2nd gen lentivirus. The packaging mix costs about $900 for a 20 rxn of 10cm plates. Contrary to their claims — the titer from 20rxn prep is not high enough and does not work with T cells.
The other system that I am using is pWPI transfer vector with pMD2.G and pCMdeltaR8.74 packaging plasmids from Trono lab thru Addgene. With this system, I am able to generate high enough titer to get a very good transduction of 293T cells but they do not seem to be working with activated T cells thou.
Both these transfer vectors have EF-1a promoter so silencing as seen with CMV in certain cell types should not be an issue. Maybe I will have to try different packaing plasmids.
Thanks
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