I am attempting to produce lentivirus vectors using the pLKO.1 system according to the following protocol: http://www.addgene.org/tools/protocols/plko/
In this protocol, HEK 293T cells are transfected with the pLKO.1 vector of interest, the envelop and the packaging plasmids. The HEK-293T media is then collected after the cells have had time to produce and release virus. The protocol gives a procedure for selecting cells that have been successfully infected with the virus, but not one for measuring or even verifying the prescence of the virus particles in the collected media (or a solution of concentrated, purified virus derived from the collected media). The pLKO.1 vector does not have a fluorescent component, to my knowledge.
So my question is, how can I detect lentiviral particles in a solution?
A qPCR for lentiviral RNA extracted from the supernant may solve this problem. This is a strategy I once learned from a Clontech product named "Lenti-X qRT-PCR Titration Kit", and similar products have not been found elsewhere. The kit provide a standard RNA sample with known copy numbers, so the copy number of your own viral RNA samples could be quantified. I guess the qPCR primer may taget conservative fragments of lentiviral genome, maybe the LTR sequences. Unfortunantely, I have little experience on this product to date, so the protocol is attached here, hope it will help.
A qPCR for lentiviral RNA extracted from the supernant may solve this problem. This is a strategy I once learned from a Clontech product named "Lenti-X qRT-PCR Titration Kit", and similar products have not been found elsewhere. The kit provide a standard RNA sample with known copy numbers, so the copy number of your own viral RNA samples could be quantified. I guess the qPCR primer may taget conservative fragments of lentiviral genome, maybe the LTR sequences. Unfortunantely, I have little experience on this product to date, so the protocol is attached here, hope it will help.
You can perform p24 ELISA assay on the HEK 293T cilture supernatant.
We use routinely the INGEN kit.
Hi Stephan,
Alternatively, you may want to use what's called a PERT (product enhanced reverse transcriptase) assay. It's actually a RT-qPCR, but you're using the reverse transcriptase content of your sample to initiate RNA to DNA conversion of a provided viral RNA sample. So the more virus, the more RT, the more DNA you end up with. If you're doing this regularly, it may come cheaper than comercially available kits.
I use qPCR kit from abm. See link here: http://www.abmgood.com/High-Titer-Lentivirus-Calculation.html. It's easy, but a little expensive.
If all you are after is simply to check whether you are producing vector, one of the easiest ways would be to just find anyone working in your city who works with HIV and ask them to test your supernatant. These guys will have established protocols and will be a great resource for trouble shooting as well
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