Problem with Lentiviral production - I have problems getting good virus titer!! (Mar/04/2011 )
Hi guys!
I know there are several topics like this, but I tried a lot of things and yet I have not good results...
I cloned my protein of interest in diferent lentiviral vectors (3rd generation vectors, about 10kb, and with puro rersistance).
I checked the expression of my protein by transient transfection in 293t and the expression is quite good, so the plasmids are working.
Now I want to produce virus!. First I thawed new 293t, I checked with a control plasmid with GFP plus the other 3 plasmids encoding the envelope and the structural elements (5ugr of each one in a 10cm plate),so I could see that the transfection efficiency was good (80-90% of my 293t were green), then I waited 48 hours I took the supernatant and infected MEF cells. After 2 days I could see some green cells, not too much, but enough I think.
Now the problem is when I try with my contructs! I produce empty vector(without GFP but with puro), and my constructs (in the same vectors w/o GFP, with PURO), and 48h after infection of MEF cells, I add puro (2-3 ugr/ml), and I can see how in the non-infected cells they are all dead, in the infected with the empty vector, there are a lot of survivals, but the cells infected with my constructs look like the non-transfected, so no survivals there!!
My gene is about 4Kb and the empty vector is 10KB. I dont know if that is a problem.
What can I do to improve the titer???
Thanks!
laurequillo on Fri Mar 4 14:26:26 2011 said:
Hi guys!
I know there are several topics like this, but I tried a lot of things and yet I have not good results...
I cloned my protein of interest in diferent lentiviral vectors (3rd generation vectors, about 10kb, and with puro rersistance).
I checked the expression of my protein by transient transfection in 293t and the expression is quite good, so the plasmids are working.
Now I want to produce virus!. First I thawed new 293t, I checked with a control plasmid with GFP plus the other 3 plasmids encoding the envelope and the structural elements (5ugr of each one in a 10cm plate),so I could see that the transfection efficiency was good (80-90% of my 293t were green), then I waited 48 hours I took the supernatant and infected MEF cells. After 2 days I could see some green cells, not too much, but enough I think.
Now the problem is when I try with my contructs! I produce empty vector(without GFP but with puro), and my constructs (in the same vectors w/o GFP, with PURO), and 48h after infection of MEF cells, I add puro (2-3 ugr/ml), and I can see how in the non-infected cells they are all dead, in the infected with the empty vector, there are a lot of survivals, but the cells infected with my constructs look like the non-transfected, so no survivals there!!
My gene is about 4Kb and the empty vector is 10KB. I dont know if that is a problem.
What can I do to improve the titer???
Thanks!
Hi laurequillo,
May i know on what basis you chose to use 2-3 ugr/ml of Puro. Have you done kill curve. If not perform kill curve. Then use that concentration it will kill all your non-infected cells.
To increase the titer. Optimize the ratio of different plasmids for co-transfection. what method are you using for transfection? Try capo4 method for 293T cells. Since you don't have GFP in your working plasmid. Do all the standardization with GFP plasmid & replicate the same with your experimental plasmid (although your gene is 4Kb you are left with no other option. I don't thing there will big difference).
I too worked with 3rd generation Lentiviral vector some time back, I used 1:1:1:1 ratio of all 4 plasmids for cotransfection, after 48hrs i harvested supernatant then passed through 0.4um filter. Titer was calculated by serial dilution followed by infection in 293T cells. After 48hrs i counted number of fluorescence. To infect my test cell line i used MOI between 10 to 100 depending on cell line. Fibroblast are difficult to transduce. they need higher MOI.
Regards,
Sen111
What kind of competent cells did you use to amplify your constructs? Your insert is very big and if your not in the right bacterial cells you'll produce empty vector but will deal with an issue of homo. recombination and your transducing vector will get messed up. Even run a diagnostic digest to make sure your plasmid is what it should be. Try using MACH1 cells from invitrogen.
Thanks guys!
Puro selection: I use the amount of puro that kills my cells in 2-3 days, so 2-3 ugr/ml.
For transfection: I use Lipofectamine 2000 which is quite good and non toxic.
Competent cells: Actually I did not think of that...but I used Top10 cells, and I checked the plasmids by normal transient transfection, and the expression was quite good, so I guess the constructs are ok!
Actually I got some surviving cells after one week with Puro, so lets see what happens this week!!!
Thanks again
I have seen something similar, although with shRNA lentiviral vectors. It has been confirmed by other people in other labs here, but it seems that for some obscur reasons, empty pLKO.1 puro give a lot more active virus then any shRNA containing pLKO.1 puro vectors. This maybe the case for your insert as well.
I currently use polyethylene imine to transfect and it works like a charm, is not toxic, and cost like 1% of the value of lipofectamine/fugene/gene juice.
People here either still use pLKO-based vectors and adjust the amount of virus per experiment, or clone all their shRNAs into pLVTHM bi-cistronic lentiviral vector.
Good luck!
Hi,
Attached is my protocol for everything I do to create high titer lentivirus. Its quite a bit different than your method but it might give you some ideas.
Shane
Thanks guys!