I am trying to generate lentiviral particles from a insert (4Kb) cloned into a lentiviral vector (pWPI, 11Kb) and I am having some issues. I transfected HEK 293T cells with the pMD2G, pPAX and our construct included into the pWPI plasmid (as usually we do it at our lab). I usually check if the cells are expressing GFP next day after the transfection and my surprise was that I obtained GFP+ cells in the controls (pWPI plasmid) but not in my construct. Does anyone now if it's possible that as this construct is large-size (about 15Kb), the expression of GFP can result decreased? I have been working with other pWPI cloned vectors and I never had this problem before. The pWPI plasmid is a IRES-GFP vector and we cloned our construct upper the IRES-GFP site. Thank you very much for your answers.
I ever had a similar problem when I cloned a 3.4 kb gene yet lentivirus still can be generated and later on selection worked. However, when I used western blot to analyze protein level I didn't see an overexpression of my gene of interest.
Length may really determines. I was sure that this pWPI-MCS-IRES-GFP is a reliable system and I cloned many genes including smaller ones. Again, by western blot I saw nice overexpression of smaller ones but only not this larger 3.4 kb gene. When you clone a 4 kb insert into pWPI, the distance between 5' and 3' LTR is larger than 9.5 kb that exceeds the natural length in wild-type HIV.
Let's wait for more answers.
4kb is especially large for lentivirus. The total lentivirus cassette (5' LTR to 3' LTR) should not exceed 10kb for optimal results. As you push beyond the upper bounds of the packaging limit, the viral titer will fall off precipitously.
However, it sounds like you are observing low GFP in your 293T cells during virus production. This is likely attributable to the fact that the efficiency of IRES-GFP expression is dependent on the length of the preceding cDNA, so the large cDNA size may attenuate your GFP reporter expression. Additionally, plasmid transfection efficiency is inversely proportional to the plasmid size, so for a 15kb vector, you probably need to increase the amount of vector DNA by about 1.5-2 fold, to get decent transfection efficiencies.
I would recommend not worrying about the GFP expression levels, and instead focus on optimizing your viral titers.
Thank you very much for your answer Bingqian Qu. I would take it in consideration!
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