Dear sir,
Because my project have to express a new receptor (CAR) on the surface of T cells, and i used lentiviral to transduce T cell.
However, in the process of producing virus, beginning with transfect HEK293T with vector carrying CAR construct (present GFP as a marker) including the component of the virus in other vectors. Although, the result of transfection presented by detecting GFP expression with flow cytometry was very good (GFP expressed about 80-90%), but when i used the virus from supernatant (after concentrated the supernatant and filtered with 0.45 um) to transduce HEK293T cell again for check titer of virus .... the result was really bad (GFP express about 10-15%)
how is it possible????? i would like to ask you why the result be like this and how to solve this problem.
Please share your experiences and suggest me to solve this problem
The filtration step of the lentivirus is the culprit for low efficiency of the virus. I suggest to do the centrifugation as sterile as possible and use the concentrated virus as it is without any filtration. In general, the virus pellet can not be resuspended well to their individual viral particles and so there will be large virus clumps which may be bigger than the pore-size of the filters used for sterilization. So using them for sterilization retains a large portion of the virus at the membrane which contributes to the loss of efficiency of the transduction. I hope this solves your problem. Good luck
Hi Thanida!
when you do a transfection you put a lot of molecule of ADN ( >106 mol) /cell. it's very very superior to your quantity of your virus in your supernatant. Make the calcul of the quantity of ADN molecule that you put per cell and compare with your quantity of viral particule. i think you will be suprise. Futhermore about me there a lot more molecule of ADN enter in the cell compare to viral particule.
good luck
have a nice day
thank you all of you, sir..
In my process after transfected HEK cell, i collected viral supernatant, then used 0.45um filter (PES) to filtrate the virus before using lentiX concentrator to concentrate the virus. i stored it at -80 degree celcius before using in transduction step in HEK.
and in the ratio of transfer vector, packaging vector and envelop vector was followed by the general protocol in the paper.
actually, im worrying about the structure of the protein receptor because i designed it by myself using many domains from many receptors to form and express in one receptor, in addition adding GFP as a marker. im not sure whether this structure is so stress or not. by the way, in the transfection process i can see the expression of GFP in high amount....
therefore, in your opinions, do you think the lower transduction efficiency will come from the structure of protein or not??
Best regards,
Protein itself have nothing in common with transduction efficiency. You deliver everything as RNAs particles. So if GFP is not So if GFP coding sequence is not fused with this Frankenstain protein then the answer is no. It is normal that you get 15% transduction rate. The question is that you say nothing of the volume of supernatant you have used etc. Just use more to get better transduction rate. Or check some articles how to increase the titer of LentiV.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus