No virus titer and MSCV-mir30 vector problem - HELP (Mar/10/2007 )
You have to check with proper restriction digests, which could indicate, if there has been changes in the LTRs. Some times, there might be sites next to the LTRs for verification.
Good Luck !!!
-scolix-
Are you using the MSCV system from Clontech? Their literature on this kind of problem is spectacularly unhelpful, so I would suggest you contact a representative of the company you got the system from and ask if it is possible to check the LTR integrity, and how you can do it.
If it is not possible, go back to the beginning and use SURE2 cells (Stratagene) for your plasmid manipulations, and these cells will prevent the recombination and mutations which damge the viral LTRs.
-scrat-
QUOTE (laska @ Mar 10 2007, 08:58 AM)
I have a problem. I have produced ( was thinking that I did) viruses in my 293T cells, and Phenix A cells. Then when I'm infecting my target cells I can not see any expression from the vector. The construct has incorporated GFP ( expression from SV40 promoter). Almost all of the packeging cells were very green, but almost none of target cells!!! What could be the problem. I inserted shRNA into my vectors, and I have confirmed the integrity of the vector, and I have also sequenced everything. The data at that point look very good. I was soo sure that everything should be fine, but now is 72h after infectionofmy target cells, and nothing they are blank like...... Please help me with that?
I have also tried infect 293T ( as target cells) and nothing..... so what went wrong between green packeging 293T and no green target cells. I'm soo worried any suggestions
I have also tried infect 293T ( as target cells) and nothing..... so what went wrong between green packeging 293T and no green target cells. I'm soo worried any suggestions
How many number of passages did the phoenix cells go through. The reason i asked is that the packaging signals are not integrated into the the genome but are incorporated on 2 different episomes. it could be possible that they are diluted due to number of passages and no drug selection of episome.
Try repackaging with the earliest frozen stock of the phoenix A cells.
more info can be found on Nolan Lab home page (google it)
You may also try to design primers to sequence the LTR region to look for mutations/ validate its integrity. (restriction digestion would be the quickest)
Good luck!!
-Sam
-Sam Suri-