I am planning to make lentiviral shRNA particles of my gene of interest for injecting into the brain. Can anybody tell me the best way to make lentiviral particles in the lab? What is the best way to concentrate them? I would be using pLKO system.
Package in HEK293T cells by cotransfection. These protocols are pretty well documented. I usually transfect 10-20 15cm dishes of 80% confluent HEK293T. For concentrating, I centrifuge the supernatants overnight at 4°C and 4000 rpm in 250 mL bottles to pellet the virus. You will likely not be able to see the pellet. On ice, rinse very gently (do not pipet directly where you think the pellet is, but swirl slowly to rinse) with PBS, then resuspend in 500ul PBS / 250mL supernatant. For viruses that package efficiently, you can get titers of >40-80 e6/ml. However, for brain injections, this may not be the cleanest viral prep. We were always doing in vitro work.
Goutam- PEG mediated precipitation works well for concentrating lentivirus. Homemade recipes exist, or you can use commercially optimized reagents/protocols such as SystemBio's PEG-iT reagent, www.systembio.com/lentiviral-technology/delivery-systems/peg-it/
If you are working in a murine system, you might have problems with lentivirus. Murine cells express a number of host factors that interfere with lentiviral infection. In general, I think people prefer adenovirus or AAV for in vivo experiments; however, I can't comment on the tropism of these viruses towards cells in the neuronal lineage specifically. Would advise a careful review of the literature to see what type of viral vector is most frequently used in your experimental system.
Hi,
Thank you very much Jason and Daniel. These are the very good suggestions. Well, Jason I am planning to use 6 well /60 mm plates for my co transfection. I wonder whether PEG mediated concentration of lentiviral will be good enough for brain infection. Daniel I have consulted many literature people has used lentiviral vector besides AAV for gene delivery to brain. So I have made my plan already. And ya I cant afford buying kit reagents so I need to follow the household recipe. I just like to ask one more question whether the orientation of the construct in the pLKO vector has any role for gene expression. I made the shRNA construct against my gene of interest in the reverse orientation as of the Scramble shRNA containing pLKO vector. thank you very much friends for your help!!
Yes, 5' to 3' direction of the sequence matters. You need your gene-targeting sequence to be in the sense orientation relative to the U6 promoter. You should test your virus in vitro on tissue culture cells to determine viral titer, and also verify that your shRNA mediates gene knockdown, before proceeding to your in vivo experiment.
Hi Goutam,
we tested two methods of purifying lentiviral particles: via PEG and ultracentrifuge and actually came to conclusion that there is no negative "PEG" effect on infection of primary human hepatocytes in culture.
You can find quite a detailed SOP here: http://seek.virtual-liver.de/sops/13
Good luck!
Hi Daniel, I am not talking about the orientation in terms of U6 promoter I am talking about in terms of LTR of the construct. I have cloned the shRNA under U6 promoter and the total cassette (including U6 promoter-shRNA-terminator) is reverse oriented in compare to the scramble counterpart. thnks!
Sorry, I misunderstood your previous post. It sounds like your constructs will work. Usually, scrambled sequences are cloned in the same orientation as the targeting sequence. Although it is good to include the scrambled sequence as a control, I think many people still expect investigators to demonstrate the same phenotype using two distinct shRNA targeting sequences. If your initial experiments work, you might try generating a second shRNA against your GOI for validation purposes.
Thank you so much Dr Daniel. It will definitely going to help me. And thank you Maria I am gonna use PEG to concentrate the lentiviral particle!!
Hi Daniel and Goutam, It was nice to read your conversation regarding Lentivirus. I am also planing in house production of lentivirus. Where can I get the protocol in detail- from start to finish, including titration. It will be a great help if I can get the answer..Many thanks in advance. Roy..
Google for Tronolab. Find the link to their Protocols. The one on their website suggests CaPO4 transfection. I've used Lipofectamine 2000 with similar success. Use whatever transfection method you have up and running. The critical aspect is to make sure you use appropriate ratios of lentiviral to packaging plasmids.
I use the 293T cells for virus production using TransIT followed with ultracentrifugation to collect the virus particle. Then I use the HCT116 cells to calculate the titer unit of virus particle. If you still need some protocol, I can show you. Cheers.
Thanks Daniel, Kewalin and Amanda, all of your answers and suggestions are helpful. I will get back if needed. Thanks again..:-)
Thanks Daniel, I got the protocol from Tronolab only. @ Swagata Type the name of your plasmid in the Addgene site it will give you the protocol associated with your plasmid type.. I got it from there only,, mine is pLKO based system. Thanks Kewalin and Amanda ...all the suggestions are very useful for the new comer like me..
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology