3 replies to this topic

[Houri]

Hi everyone,

 

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.

[CPRES]

-You get the titer from viral genomic RNA, which would not exist unless the viral was packaged properly.

-WPRE is located in the 3' UTR of the transgenic RNA, so you will not get the titer from some empty virions not expressing your transgene.

-10.2 Kb is right there. I have worked with smaller ones, but know many people who have used 10 kb cargo in lentivrus with equally good production and transduction.

 

I would focus on making sure your are transducing them right. But beyond that, dunno.

[gfischer]

In my experience, transduction can become less efficient with larger transgenes, so that may be a factor.  Are you using a polycation in your transduction (i.e. Polybrene)?

 

Also, HEK cells should tolerate an MOI higher than 20, is there any chance that your transgene is toxic to the cells?  Do you have another cell line you could try the virus on, like HeLa?

[Houri]

Thanks for the replies.

no I haven't tested polybrene yet, but I will. This virus will ultimately be going in vivo into the brain so I just wanted to test it without additional factors that I will not be injecting in vivo. Also my transgene, only expresses in the presence of Cre whereas RFP expresses in the absence of Cre (it's conditional) so I should be able to see RFP at least even if the transgene is toxic, in cre negative cells that is. And because the HEK cells tolerate the entire insert well when it's expressed from a plasmid, I'm assuming the insert is tolerated and it seems the viral solution is toxic rather than the genes? 

The cultures started lifting off after 16hrs, so I changed the medium and they recovered. Would you recommend a shorter incubation time with a higher MOI? Do you have any thoughts on the FBS in the medium? I'm keeping it in there, although I know some protocols suggest excluding it. 

 

 

Thanks!