I have been working with 3rd generation lentiviral vectors. The problem is that the transduction efficiency is very low and GFP signal is expressed atleast after 72 hours (although the expression is under the control of CMV promoter).
And in this duration normally cells take atleast 2 divisions and then the population for sorting becomes more difficult to analyze and sort through FACS.
Is there anyother way to select transduced cells or I can start with low cell density and high viral load without doing titre and then sort them through FACS. Any suggestions please.
If I just need some transduced cells, which I will then sort and make a new cell line, I generally won't bother to estimate the viral titre, since the exact transduction efficiency is not important. I will just transduce a small number of cells in a small volume (e.g. one well of a 24-well plate) and use a lot of viral supernatant (e.g. 100-300 ul in a 24-well plate). If the cells become too crowded before you are ready to do FACS, it is perfectly fine to split them first e.g. I might scale up from a 24-well plate to a 6-well plate before doing FACS.
If the transduction efficiency turns out to be quite low (80%), then many of the cells may have more than one copy of the transduced gene. If this is a problem for you, you may wish to repeat the transduction with less viral supernatant. But for most purposes, a high transduction efficiency and multiple copies won't be a problem if the cells are still healthy.
Thanks SHanshan. Yes the efficiency is very low and I have the same options in mind as you have suggested but is there any criteria or cut off to avoid any toxicity situation if I increase the viral load.
I read somewhere that the viral supernatant should not make up more than 30% of the liquid in the well. Did you use polybrene and spinoculation (I spin the plate at 1200 x g for 1 h)?
No I havent. But I think its a good option. I will try this. Thanks for help
Dear all
I think the titer and/or the purity of your vector batch is not enough high to allow a good transduction efficiency. Clontech provides very efficient ready to use highly concentrated GFP expressing-lentiviral vectors. An antibiotic resistance gene is included inside the expression cassette for cell selection.
Please have a look an the following link:
http://www.clontech.com/FR/Products/Viral_Transduction/Lentiviral_Particles/Fluorescent_Proteins?sitex=10023:22372:US
Pascale
It sounds like your virus titre or your GFP expression is suboptimal, which will make things difficult for you. Normally, you can see some fluorescence after 48 hours and it's brighter by 72 hours.
What kind of transfer plasmid are you using? How big is your transgene? What cell type are you trying to infect? How does GFP expression look when you infect 293Ts?
Hi ill,
Previously i used 20 ul of the concentrated virus for transducing 12 well plate and the efficiency was very low. After increasing viral load to 500 ul of the concentrate I got good signals after 48 hour that enhanced after 72 hours.
pSicoR is my transfer plasmid and I am doing transduction experiments on MCF-7 and a myeloma cell line RPMI-8226.
The expression of GFP after transfecting HEK was exactly the same as is reported due to the expression of VSV-G envelop protein. Like Over expressed all over the cell.
500ul of concentrated virus sounds like a LOT. Ok, after you produce your virus, add 1ul of virus to one well of a 24-well plate containing 293T cells at 50-70% confluency. After 2-3 days, count the number of green cells. You can then work out your virus titre. If you don't know your titre then you don't know where your problem might be.
Please correct me if I am wrong but I read in the technical pages of invitrogen and some other companies that transducing (not transfecting) cell line containing SV40T (like you mentioned HEK293t) , IS NOT RECOMMENDED AT ALL. this is because of their SV40T as it initiates unnecessary viral replication which gets terminated because of the LTR deletion making it transcription deficient and somehow cells die. So titre estimation in these cells may not be a good option.
however one can try HEK293 only as they are easy to transfect and transduce.
In my case I have transduced MCF-7 with 500 ul of viral innoculum and my cells are working perfect. Now I have passaged them and waiting to get good number to sort them.
Thanks for suggestion.
No, in my experience 293Ts transduce just fine. I have never seen any cell death even when I transduce at very high MOIs. The SV40 T antigen can stimulate endogenous retrovirus transcription but whether that affects LV transduction or performance is anyone's guess.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus