No virus titer and MSCV-mir30 vector problem - HELP (Mar/10/2007 )
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
Have you verified the other plasmids that you use for cotransfection for virus preps? Check them by running it on the gel and try analytical digest to confirm their identity. If you think some thing is a problem, you might have to redo the maxi preps.
Also I hope the cloning plasmid was grown in bacteria which prevent recombination. This could as well interfere with virus preps.
Hi!
Thanks for suggestions. I'm not sure about the bacteria the plasmid was grown in. Anyway with Phenix A cells you do not do cotransfections , I'm only addeding my retroviral construct. The cells have all machinery for gag, pol, env etc. I was thinking what about the medium, you grown cells during transfection, and the presence of antibiotics in it. Can it make any difference if D-MEM is supplemeted with P/S or not?
OK, I'm not sure if I've understood you correctly, but you are saying that you get GFP expression after putting your plasmid into the cells, but then no virus is being made? MSCV is a retrovirus system, is that right?
If I've got that right, I think what is happening is that your plasmid is damaged. Does it have long terminal repeats? How did you check vector integrity? If the plasmid is damaged/mutated, you will still get GFP expression in your cells because thats under the control of the plasmid promoter. But, the plasmid will be unable to produce any virus.
I've seen this happen with both the AAV (inverted terminal repeats) and lenti-viral (long terminal repeats) vectors we use in this lab.
I do transfections in DMEM, 10% FBS and no P/S, as recommended for lipofectamine. Change media after 12 hrs to normal media with P/S.
i still beleive you should try to verify if your plamids have undergone recombination or not and to use a recombination negative bacteria for growing them.
If I've got that right, I think what is happening is that your plasmid is damaged. Does it have long terminal repeats? How did you check vector integrity? If the plasmid is damaged/mutated, you will still get GFP expression in your cells because thats under the control of the plasmid promoter. But, the plasmid will be unable to produce any virus.
I've seen this happen with both the AAV (inverted terminal repeats) and lenti-viral (long terminal repeats) vectors we use in this lab.
Hi!
Yes, no virus but many very bright green fluorescence cells. MSCV is a retrovirus system. I have checked the vector integrity by agarose gels. I was also thinking that maybe something happened and the plasmid is damaged, by then is it a metter of cleaning the plasmid for example some GFX columns, or I have to go to the begining and start all the cloning yet again from there. I was sequencing the plasmids both mini and midi preps and it was fine, even better for mini preps. So what you will do if were me. I'm sorry but I'm new in that field, and for me is just the tool I need to make for the future experiments, so I have to do it as fast as possible. It took me a lot of time to make 14 different MSCvs ( different linkers insered) and would love to save my work and time, and do not go back to begining, is it even possible?. Thak you and wish you nice day.
Magdalena
i still beleive you should try to verify if your plamids have undergone recombination or not and to use a recombination negative bacteria for growing them.
Hi!
I have changed the medium for normal complete medium, but still nothing. I'm very new in that field, so I'm sorry for asking but how I could check that the plasmid has undergone recombination? for me is just the tool I have to make for my further application, and it drives me crazy. I spent soo much time making 14 different vectors ( different linkers in my MSCVs) and now cant get any viruses!!!! Is there any way to save my samples and time. I have confirmed sequence. Ok is it possible that I will have still GFP expression since that is from plasmid promoter, but no viruses will be make?. And if you make in your packeging cells transfection, and for example expect to see silencing effect from your shRNAs, could you see that, or you can only get that in your target cells, from the viruses?
thanks
i still beleive you should try to verify if your plamids have undergone recombination or not and to use a recombination negative bacteria for growing them.
Hi!
I have changed the medium for normal complete medium, but still nothing. I'm very new in that field, so I'm sorry for asking but how I could check that the plasmid has undergone recombination? for me is just the tool I have to make for my further application, and it drives me crazy. I spent soo much time making 14 different vectors ( different linkers in my MSCVs) and now cant get any viruses!!!! Is there any way to save my samples and time. I have confirmed sequence. Ok is it possible that I will have still GFP expression since that is from plasmid promoter, but no viruses will be make?. And if you make in your packeging cells transfection, and for example expect to see silencing effect from your shRNAs, could you see that, or you can only get that in your target cells, from the viruses?
thanks
Could you try a analytical digest such that you could verify the presence of terminal repeats etc, as these might be the regions which undergo recombination.
Also did you try using different amounts of viruses, try adding lots of viruses just so that you know if its working or not.
What are the target cells? Will your promoter work in these target cells? You need to check these things.
Verify all plasmid are fine by digest and running on gels. and I would suggest you to try to infect all 3 plasmids for virus preps in the 293T cells. And using the supernatant from this transfection to infect your target cells.
The packing cell lines lose efficiency as they are passaged so you could try get low passaged Phoenix cells for virus prep again.
good Luck !!!
Hi,
Ok, a few more questions. What E. coli strain did you grow your plasmids in? When you say you ran the plasmids on a gel to check integrity, did you digest them with restriction enzymes to test if your LTRs are still OK? (I'm not sure if this is possible with the MSCV system).
It does sound like you may have lost your LTRs on your plasmid, and if this is the case I'm afraid you will have to re-clone 
Don't worry, I did exactly the same thing with an AAV plasmid!
Ok, a few more questions. What E. coli strain did you grow your plasmids in? When you say you ran the plasmids on a gel to check integrity, did you digest them with restriction enzymes to test if your LTRs are still OK? (I'm not sure if this is possible with the MSCV system).
It does sound like you may have lost your LTRs on your plasmid, and if this is the case I'm afraid you will have to re-clone
Don't worry, I did exactly the same thing with an AAV plasmid!
I checked the plasmid integrity just by runing it on the gel. No digestion. I sow very nice and clear band for my circular form, then small for linear and naked plasmid as well. Even as I wrote the band seams to be samller in the size, which worried me a lot. That may suggests that there is something wrong with the vector. How can I check then with MSCV system that I have lost LTRs?