For GFP labelling, I am using P201, VSV-G and Delta 8.9 for the transfection along with PEI in the HEK 293T cells. I used the Qiagen kit for the isolation of plasmids as per the protocol. The construct worked after a week i.e., I observed the fluorescence in the HEK293T cell line. Assuming that the virus packaging also worked, I infected my culture of interest with the supernatant (filtered). After 48hours of transduction, I was not able to see any of the cells tagged with GFP. Could you please suggest as to what should be done in order to achieve proper packaging of lentivirus or what could be done to troubleshoot the same?
@sowmya ramesh.. 293T cells are very easily transfected and the p201 plasmid, if it is what you expect it to be, then you should be able to see GFP expression in as little as 12 hours. From your initial transfection, I would think that there is some problem with either the transfection reagent (PEI) or with the plasmid itself.
For the plasmid, you could perform a PCR for the presence of CMV-GFP and/or a diagnostic restriction digest and confirm the accuracy of the plasmid.
As for the PEI, there is a very specific way to make it and you cannot make the transfection reagent with any run of the mill PEI available. It has to be a linear unbranched polymer of at least 25kDa, which you have to dissolve in distilled or ultra pure molecular biology grade water.
The other way to confirm your transfection is to observe for syncytia in your transfected cells. VSV-G would form massive syncytia (huge cells with multiple nuclei) in most cell lines if transfected efficiently.
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