Hi Matteo,
First question: Why do you want do this with Hela cells and not 293T? An other way possible?
If you have to do it in hela cells, you have to generate a special lineage of hela: which constitutively expresses the HIV genes essential for production or which contains the SV40 origin of replication to allow the replication of transfected plasmids.
Hello Nicolas,
thanks for your interest. I need to produce virus from HeLa as a control, I am already producing it from 293T cells. I am using 3 plasmids, one coding for Gag/Pol, one for VSVg and one for the genome. I would like to implement the same protocol in HeLa cells to get a vector.
Ok, so i think you could look for how transformed hela cells with a plasmid containing the origin of replication of SV40 to have the same protocol of production (tri-transfection). However your vectors will not have exactly the same envelope that vectors produced by 293T, indeed the cellular membrane is not exactly the same between hela and 293T. Could you not generate your control by another way?
One of the major blocks to production of virus from Helas is expression of Tetherin/BST-2/ HM1.24. You absolutely require the HIV-1 Vpu protein in your transfection mix if you need to produce your virus in Helas. What is the Gag-Pol construct that you use? and does it have the coding region for Vpu intact?
The other problem is transfection efficiency. If you use the appropriate transfection reagent for Helas, you will be fine. But definitely check for Vpu presence. Alternatively, several viral proteins from different viruses have been found to antagonize tetherin. You could use any of them in your transfection to produce enough viruses.
Hello Sanath, thanks for your reply. Do you have a reference for Tetherin and Vpu in HeLa? That seems interesting!
http://www.ncbi.nlm.nih.gov/pubmed/18200009
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2844890/
Hi,
If you didn't see this paper: http://jvi.asm.org/content/73/2/887.short
They did lentiviral productions in hela cells but the M&M is not very detailled. However you could contact some of the authors...
and I would add:
thanks Sanath for your insight about vpu. However, I think even with a very efficient transfection method it will be very difficult to have a good production. I think you have to perform a system which will allows the replication of transfected plasmids (as SV40) or generate a hela cell line which constitutively expresses production factors.
Thank you everyone for the inputs. I knew about tetherin and I thought of it as a possible problem. I will check the protocol on the Journal of Virology paper and see if I can get some infos. I actually have a SV40 origin on my Gag/Pol plasmid.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology