About stratagene's Helper-free system - (Aug/31/2006 )
do any guys here use stratagene's helper-free system?
I use all of its plasmid: pAAV-MCS(EGFP was clone in the MCS),pAAV-RC, pHelper.
The only difference is the package cell, i use HEK293 instead of the recommended AAV293.
My transfection is successful, but my 4 times of transduction all failed.
I strictly stick to the protocol of the manufacturer.
An odd phenomenon is, when i view the 48hr-post-transduction cells under a fluorescent microscope, none of the cells could be seen green fluorescence, but a strong green background can be seen, after i change the supernatant to fresh DMEM, the strong green background turned very weak or totally dark.
I don't know why, Who can explain and troubleshooting for me? thanks!!
use the AAV293 cells for virus production. u will get 10x more virus with that than with the normal HEK293.
What do u mean by "my 4 times of transduction all failed". Do u mean that after infecting with the virus, u dont c any green fluorescent cells?
AAV takes longer than other viruses to express so give it more than 48hrs unless u r using AAV-1 serotype. Also do ur cells express the right receptor for AAV and also the right promoter. These can make differences in the AAV.
Which serotype r u using ? 2 ?
What do u mean by "my 4 times of transduction all failed". Do u mean that after infecting with the virus, u dont c any green fluorescent cells?
AAV takes longer than other viruses to express so give it more than 48hrs unless u r using AAV-1 serotype. Also do ur cells express the right receptor for AAV and also the right promoter. These can make differences in the AAV.
Which serotype r u using ? 2 ?
Thanks a lot! They are very valuable information. yes i mean i don't see any green fluorescent cells after infecting
i use 2.
Another question, how do you say "use the AAV293 cells for virus production. u will get 10x more virus with that than with the normal HEK293" Any evidence? your experience or literature claimation?
And I wonder how virus show under a fluorescent microscope if they express the EGFP protein.
Thanks
We used to make AAV in our lab. And the titres increased when we switched from 293 to AAV 293 that stratagene provided.
under microscope, a infected cell will glow green if GFP is present. Its notm uch different than after transfection (the intensity might b lower with viral infected ones compared to transient transfection)
Thanks