TRIPZ lentiviral production - problems with viral production (Mar/30/2011 )

Hi

I am trying to make lentiviral particles with TRIPZ from Open biosystems. I'm having problems meaning I am not being succesful at infecting cells. I don't have anty problems with other viral vectors such as pLKO.1 or expression lentiviral vectors and I am using psPAX2 and pMD2.G as packaging. They should be OK since they are II generation asOpen biosystem recommend, but still, no success with TRIPZ. fugene6 is the stuff I use to transfect 293FT. I figured the size of the TRIPZ (13Kb) might be a problem. Does anyone have any suggestion?

Thanks very much

Barbara

Hi,
I have a similar problem - I tried producing pTRIPZ-derived lentiviral particles using pCMV VSV-G and pCMV d.R8.2 dvpr (2nd generation system from addgene) that work perfectly with pLKO.1 and did not get an any virus. Now I am wondering what went wrong. The tat gene should be included on pCMV d.R8.2 dvpr...
Did you get the pTRIPZ lentivirus production to work? What system did you end up using and did you find out what went wrong initially?

Thanks very much

Michael

13 kb is quite large. I would recommend modifying your transfection protocol or trying other transfection reagents, though fugene6 is one of my favorites.

In my hands Trans IT 293 from Mirus works perfectly for packaging lentivirus. You may want to try it. They ship you a free sample of 100ul and you need to use it for the ratio of 1:3 (DNA:Reagent)

Ambinlab thanks for the suggestion. I have had a similar problem with Tripz vectors, no virus production from any of 3 different vectors bought in. I even tried to express the vector the old fashioned way (lipofectamine 2000 transfection) into my target cell line followed by puromycin selection, as the guys at open systems said that should work if I wasn't insistent on utilizing the lentiviral vector to obtain a stable cell line. I appeared to select colonies over the first week or so (which I imagine was only transient expression as they all eventually died out). I am somewhat relieved to see that I am not the only one struggling with this vector and I'll try your suggestion.

Hey. I have been really successful in producing TripZ constructs. Basically I follow a Trono Lab protocol and use CaCl2 transfection protocol. It works amazingly well for something relatively archaic and it allows me to transfect 15cm dishes. I also use the packaging plasmids psPAX2 and pMD2.G.

18 hours after transfection I change out the media to a LONZA Ultraculture General purpose serum free media that I have added NaPyruvate and L-glutamine to (virus doesn't like FBS). I put the plates at 32oC 5%CO2 incubator and harvest the media once a day for 3 days (my best yields come when I harvest the media once in the morning and once in the evening placing the media at 4oC.) I filter the media through a 0.44u filter and concentrate either by ultracentrifuge and careful resuspension or I concentrate in a millipore 100KDa Ultracentrifugal filter device that I spin at 3,000rpm until I have concentrated everything into ~0.5-1.0mL/15cmdish.