Invitrogen's BLOCK-IT lentiviral kit - problems getting virus (Apr/20/2009 )
We've gone through a bit of hell trying to produce miRNA-encoding lentivirus using Invitrogen's BLOCK-IT kit. The vectors we've been using encode GFP so we can track expression. When we transiently co-transfect the various vectors to produce viral particles, the transiently transfected cells show a strong bright green signal. However, when we collect supernatant and try to infect new cells, we see no GFP signal at all. Invitrogen suggested that our initial vector's promoter (CMV) was not suitable for our cell line, so (like fools?) we bought a new vector driven by a different promoter, but got the same negative result. Any ideas would be appreciated.
What cells (human or mouse, primary or cell lines) are you trying to infect? Have you done titration of the viral particles?
pcrman on Apr 22 2009, 02:12 PM said:
We are trying to infect both human HEK293FT (supplied by Invitrogen) and the mouse EC cell liine P19. Since my first post, I must confess that we now see some cells expressing GFP, albeit weakly. Till now we couldn't begin to think about titering because we couldn't see any signal.
We also have problem getting high stable expression in mouse cells by infecting with lentivirus. You need to do titration to determine whether the packaging is successful or not.
I have ben working with the Invitrogen Block It for some time. I have modified the vector and it actually gives a very good titre. We initially had to optimise stuff to get good titres. This is what I did.Try out cut out unnecessary elements from the vector. Donot use invitrogen's helper mix, instead make it up from their mixture. This already gives a much higher titre.
We have been using the same CMV promoter and it works fine.
1. The BLOCK-iT kit might still provide pLenti6 vector which has no cPPT and WPRE on it. The virus titer is 5-10 folds lower.
2. The att sites for Gateway recombination make the lenti vector unstable and may reduce the expression and virus titer.
3. The CMV promoter is good but might not be a good idea for long-term expression in mouse ES cells. It may be silenced after two weeks.
4. The packaging mix (pLP1+pLP2+pVSVg) is pretty good, but it's a bit tricky on transfection and transduction. You might want to do spinoculation for ES cells.
5. It looks like you are expressing miRNA precursors by using BLOCK-iT vector. You will not be able to tell the miRNA expression by using GFP since they are expreesed by two different promoters.
This web site is still under construction, hopefully you can get some ideas.
http://biosettia.com/php/02-lenti-mirna-ex...uct-description