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problem with lentiviral expression of protein


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#1 SHERRYCOUS

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Posted 10 January 2010 - 09:13 PM

Hi Everyone:

I have a problem in producing lentivirus which can correctly express the protein.

The vector I used is pLenti6/V5-DEST, and I am able to detect the protein expression if I do transient transfection and IMF using 293FT cells. I get bright specific staining.

But If I use the same plasmid and 293FT cells as packing cells, and follow the standard protocol used for virus production and collect the virus after that. Then I used the virus I get to infect 293FT, Hela or other cell lines, no targeted protein expression can be detected.

I try to measure the titer(that is I just infected Hela cells and using antibiotics selection to do the titering, many colonies were formed. ) So maybe the problem is not coming from the low-titer of virus and unable to infect cells. 

I have repeated the whole virus-production->infection->protein expression check by IMF several times and still cannot detect the protein expression. What could be the problem? Any suggestion?



Edited by SHERRYCOUS, 10 January 2010 - 09:15 PM.


#2 gfischer

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Posted 11 January 2010 - 10:27 AM

Hi Everyone:

I have a problem in producing lentivirus which can correctly express the protein.

The vector I used is pLenti6/V5-DEST, and I am able to detect the protein expression if I do transient transfection and IMF using 293FT cells. I get bright specific staining.

But If I use the same plasmid and 293FT cells as packing cells, and follow the standard protocol used for virus production and collect the virus after that. Then I used the virus I get to infect 293FT, Hela or other cell lines, no targeted protein expression can be detected.

I try to measure the titer(that is I just infected Hela cells and using antibiotics selection to do the titering, many colonies were formed. ) So maybe the problem is not coming from the low-titer of virus and unable to infect cells. 

I have repeated the whole virus-production->infection->protein expression check by IMF several times and still cannot detect the protein expression. What could be the problem? Any suggestion?



What coat protein are you using? If the pseudotype is not suitable for the cell lines you are using, you won't get transduction. The same applies if you're using a cell-specific promoter.
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#3 LavenderBerri

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Posted 12 March 2010 - 01:23 PM

Hey SherryCous ~
I believe that system that you're using the Invitrogen one right? Not sure exactly how you're doing your transductions but this is the way I've been doing mine.
PEG precipitating the virus will help to bring your MOI up which could be a problem for cell lines with low transduction efficiency. I resuspend the viral pellet in cold DMEM without FBS but with HEPES (I use 293FT as a packing line). Also, sometimes decreasing your cells/well to 5000 can help to increase MOI if this is needed.

I've been doing my transductions in a 96-well flat bottom plate with 100uls of virus (flat and not round bottom since it's important for the virus to be able to access the cells and surround it increasing the liklihood of integration. If it's a round bottom, some of the cells on the bottom don't see virus) and titrating starting with neat viral supernatent with 1:2 dilutions all the way to 1:32. This way you can try to hit the right MOI. Sometimes too high of MOI will kill cells.

Use polybrene at 8ug/ml. Incubate at 37C overnight. Change media to the proper growth medium and incubate for 48 hours at 37C. Then put into selection (I think yours is blasticidin?) by splitting into 6-well culture plates (I've been using 4mls selection media/well). However, if your cell line is sensitive to being too sparsely split, spike in your selection into the 96 well plate to get the cells to be a bit more dense before splitting (add in 10uls of 10X selection media). Leave it selecting in the 96 well plate until you start seeing cells out growing. Then split into a 6 well. If you really want to baby sensitive cells, include about 40-50% conditioned media. I found that this helped dramatically with picky cell lines.

It's a good idea to buy some commercial GFP virus to make sure that it's your virus and not your cell line that's being difficult. I've been using Cignal's GFP control. I do this routinely with new cell lines doing my transductions side by side with in house generated virus and control GFP. If the Cignal virus works and the in house doesn't, it means that the cell line is transducible but that the in house virus has a problem (not high enough MOI, not pure enough virus, etc). If the control doesn't work either, something's wrong with the protocol or your cell line is just an extra difficult one (I haven't come across a cell line yet that can't be transduced).

Good luck and let me know if any of this was confusing. =)




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