Invitrogen's BLOCK-IT lentiviral kit
Posted 20 April 2009 - 06:57 PM
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.
Posted 21 April 2009 - 10:12 PM
Posted 22 April 2009 - 07:02 AM
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.
Posted 22 April 2009 - 07:13 PM
Posted 03 May 2009 - 02:04 AM
We have been using the same CMV promoter and it works fine.
Posted 21 May 2009 - 09:27 PM
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.