Dear all,
I've been struggling with a problem regarding lentivirus production for quite some time now and I cannot seem to figure it out. I. The goal of my experiments is to knock-down certain genes using shRNAs that will be introduced using 2nd generation lentivirus. For my lentivirus, I use PAX2 and PMD2.G. The problem I am currently facing is that for some reason I am not producing any (functional?) lentivirus after transfecting the three plasmids into my HEK293T cells. What bugs me is that using plasmids that have been isolated before I joined my current lab do work fine. Here is a breakdown of my protocols and what I've tried to troubleshoot already:
Plasmid isolation:
-pLKO.1 plasmids containing shRNA sequences are grown in D5alpha competent cells
-Isolation is done using an alkalysis method that yields functional plasmid
-Restriction digestion with different RE give expected results
-After running the plasmids on a gel, most of the plasmid is supercoiled, little RNA contamination (No 18s/28s, little to no smear)
-A pLKO.1 plasmid with GFP insert is expressed after transfection in HEK293T cells (control to see if plasmid gets damaged in any way during isolation)
Plasmid transfection
-Ratio of plasmids is 4.5 : 1 : 5 (PAX2 : pMD2.G : pLKO)
-Plasmids are transfected using PEI in HEK293T cells and this is efficient since functional lentivirus can be made using other plasmids)
-as mentioned, GFP expression is observed, suggesting that plasmids are functional
-transfection is left overnight and fresh media is added to the cells the next morning
Viral harvest
-24h and 48h after fresh media has been added to the cells
-Viral supernatant is filtered using 0.22 um filters and immediately frozen down at -20C before transfer to -80C
Assessing viral functionality
-1e6 HEK293T cells are seeded and transduced with 0.5ml of viral supernatant supplemented with 10ug/ml polybrene (this is virus from the 24h harvest after filtration and has not been freeze-thawed)
-24h post-transduction the media is replaced with fresh media containing 2ug/ml puromycin (pLKO.1 selection marker) and left over the weekend. On Monday cell viability is assessed through the microscope. We do not establish the titer for the virus since we always used to get very high titers after 24h of virus production
To repeat the current problem I am having:
-the plasmids I isolate are functional and can be transfected in HEK293T cells using PEI
-the plasmids that I isolate do not yield any (functional lentivirus)
-However: Plasmids isolated before I joined the lab do result in functional lentivirus
So far I:
-Have tried plasmid isolation with the Thermo GeneJet plasmid miniprep kit
-Replaced all reagents in my isolation with fresh ones
-My supervisor has done the same experiments with me in parallel, yielding negative results as well
-Used fresh HEK293T cells
-Isolated fresh PAX2 and pMD2.G plasmids from our glycerol stock (same problem persists)
-transduced HEK293T cells with both 24h and 48h of viral supernatant without any luck
-Funnily enough, I did this using the same alkalysis method, and these plasmids work fine
-HEK293T cells are puromycin-resistant after transfection
Could somehow the PGK promotor of the puromycin-resistance gene be silenced in the process (after) transduction?
I would love to have your insight, thank you in advance!
Dear Jelmer,
This is a problem that tend to happen from time to time with pLKO based vectors. The most likely explanation is that lentiviral backbone recombination is happening. Check by restriction digestion if your lenti-backbone are intact (they tend to recombine by excising a portion of the backbone while retaining the insert). This means you'll see a GFP (by transfection) but the vectors won't be packaged.
Recombined vectors will miss one or more restriction enzymes sites, and they usually collapse to a size of about 3 kb (when linearized). If recombination is happening you'll have to restart cloning and perform it Stbl2/3 E.Coli at 30 C to avoid future recombination.
Of note, expression of eGFP or short-hairipin RNAs upon transient transfection will be often fine on recombined plasmids, but they won't be assembled (probably missing some key regulatory region as one or both LTRs).
In parallel check on gel also the integrity of your packaging vectors :) You never know!
Hope it helps and good luck
Francesco
Dear Francesco Aulicino
Thank you for a swift reply. I have checked the integrity of the packaging vectors as well with RE and they seem to be fine and are functional with older pLKO plasmids. I have also ran my pLKO plasmids of interest on gel and I have seen that for one of them indeed a part of the backbone is excised, the other plasmids seem to be intact however. Your reply did give me some insight so I thank you!
Dear Jelmer Faber,
depending on the kind of recombination that is happening you might observe seemingly intact plasmids. That's why is perhaps best to check them with a broad panel of restriction enzymes. If for at least one of them you're already observing a deletion, you're perhaps on the right track. Since all of them are not working, the others might just have micro-deletions you haven't spotted yet. Switching to Stbl2/3 (but more generally just plating and growing at 30 C) will help keeping them intact and hopefully you'll get your viruses soon.
Cheers
Francesco
I think you need more details on your viral harvest. how are you doing it? how are you spinning down and processing the media? you might be losing virus during isolation.
Dear Francesco Aulicino Thanks again for the reply! I will see what REs I can select.
Julie Ann Dougherty I have added this to my question, but to sum up: Viral supernatant is filtered (0.22 um) and directly added to my cells or frozen down at -20C before transfer to -80C
I thinks it's very likely you need a concentration step and your virus is too dilute. 0.22um might be small, perhaps try 0.45um just to be safe.
you can concentrate by ultracentrifugation:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865973/
or using a reagent:
https://www.takarabio.com/learning-centers/gene-function/viral-transduction/lentivirus/lenti-x-concentrator
Determine your viral titer then use and MOI of at least 10 to transduce cells. test in a series of MOIs as too high might overload the cells with virus and stress them.
https://www.cellbiolabs.com/lentivirus-quantitation-titer
also, spinoculation can help get the virus on the cells as well. Spin in plates at 800x for 30min to 1 hour at 25-30C.
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