I sent a lentiviral plasmid out to a company to be packaged. My insert size is very large, 5'LTR to 3'LTR size is 10.2Kb. I got a titer of 5x10^8 estimated by qPCR of the WPRE element. Transduction in HEK cells yielded just a few positive cells (RFP positive) at a MOI of 20. Higher than that kills the cells. The plasmid from which the virus was made expresses RFP and my protein of interest well in HEK cells so I know both the plasmid and the virus (if successfully transduced) express the proteins of interest. What I can't figure out is why I only have a few positive cells despite the relatively high titer of virus? Is it possible that the titer also includes virions that have not packaged properly for whatever reason? The virus was packaged with VSV-G coat protein and I have an almost identical virus (but with a smaller insert size) as a positive control so I know my cells can take up the virus well.
Does anyone have any experience with overcoming this? Would a higher titer of virus help at all or do you think it just isn't packaging well? I'm just puzzled why I get a relatively high titer but no infection!
Many thanks for you help.