I am making lenti with the lentivector pLVTHM using packaging vectors pCMVD8.91 and pVSVG. We have been successful previously with our labs protocol but lately we seem to be having problems with the packaging.
Transfections go very well with ~80% cells expressing GFP (HEK293) and I collect viral supernatant 48 and 72h post transfection.
I use a mix of polybrene and chondroitin sulfate during the concentration (30min 10,000g 4C).
I resuspend the virus-pb-cs complex pellet in 300ul of cold PBS (three 10cm plates per virus) and freeze at -80C.
I have tried to infect ES cells and HEK293 cells with the virus with no luck so far. The thing is that when I look down the microscope after I add the virus to the cells I can see spheres which I think may be the virus-Pb complex. These spheres are GFP+ in wells where I added a lentivector with GFP and GFP- in wells where I added a lentivector without GFP (e.g. PURO)
This might be a silly question but I was wondering if the virus can actually express your lentivector cDNA (e.g. GFP)? If so, would this indicate that the lenti has been packed ok but I am having problem with transduction?
Any suggestions would be of great help.
Thanks a lot.
Hi Patric. Virus suppose not expressing your lentivector cDNA. They do not have got the system but depend on their host cells. Lentivirus only packed in the RNAs transcribed from your vector with the packaging proteins to form the virus you wanted. Thus, I think that these GFP+ spheres are not virus-pb complex but may be cell debris of 293 cells. Did you measure the virus titer during your experiments? This may be helpful.
Hi Zhiqiang.Thanks for the feedback! well I tried to do this in my HEK cells. adding different amounts of the virus in but could not get any GFP possitive cells. only these spheres. Is it possible that if is very highly concentrated you don't get any infection? do you concentrate virus using polybrene-chondroitin sulphateor without? do you know what works best? I say this because when I add virus to the wells I see some sort of dark debris attaching to the bottom of the 6 well plate. I think this is the polybrene but I am not sure. if it is it may be toxic for the cells so I was just wondering if ultracentrigugin the virus longer with no pb-cs gives the same results. Thanks a lot.
I did not have experience in ES cells but tumor cells. Usually I harvest virus beginning from the next day after transfection and last for 2-3 days (3-4 times at an 4-6h interval for each day) till the confluence of 293 cell is close to 100%. Each time when I harvest the virus, I filter (either 0.22um or 0.45um filter is OK ) the medium, add polybrene (no chondroitin sulphateor) then infect target cells directly with the medium, but I will not leave the target cells in virus overnight but chang into fresh medium. After 2-3 days' infection, select the cells with puro or other antibiotics.
Since your system have been worked successfully in your lab, you would better sequence the packaging plasmids to make sure that the right plasmids was used.
Good luck.
Hi Patrick, I am having similar issues. Did you get the transduction of your target cells to work?
Hi NIcole, I made three lentivectors all based on the plvthm backbone. One had gfp in, the other puro and the another puro 2a gene of interest. Fortunately my puro and puro2agene of interest worked although not at a great efficiency, about 30% transduction efficiency when before I just to get 80%. I was just surprised about not obtaining gfp virus when it was the same backbone plasmid and alldone the same way. I also found that if I spin down my virus before transduction I get rid of the debris that I observed on the plates following adding the virus to the cell. I'm convinced this sort of black debris is the pb-cs complex. I was wondering if I could avoid using pb-cs to concentrate and instead spring for longer at a faster speed. Concentrating with pb-cs also made the virus hard to resuspend, had to pippete up and down over 20 times which I'm afraid it may have killed some virus. I also was wondering if leaving the pellet soaking in pbs at 4c overnight improves getting more virus fRom the pellet or not.
What do you think?
Not sure whether you have figured out or not. Just a suggestion. I usually infect 293T cells not HEK293 since 293T stably express SV40 large T which can bind to SV40 enhancers (carried by your lentivector) of expression vectors. The presence of T-antigen in 293T cells could help in episomal maintenance of SV40 origin containing vectors.
For viral production or other transfections 293T is now preferred since they are normally better transfectable.
Hope this late suggestion is helpful.
Hi Chen! . I did mange to solve the problem. I was using 293T not the 293s. Thanks for the help.
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