2 replies to this topic

[minemin]

Hi folks,

I'm trying to transduce hematopoëtic stem cells with a lentivirus. Producing the lentivirus is OK. But the transduction is not efficiënt enough.
Is there any possibility to make a more efficiënt virus? Or to make the transduction better?

[calvin*]

You can try increasing the transduction efficiency:
1. If you use HEK293T cells for making virus try using very early passage 293Ts.
2. If you are not concentrating the virus by ultra-centrifugation then try atleast to double the concentration by decreasing volume of media on 293T. (You can also try PEG precipitation).
3. The target cells should be in the best stage of their health.
4. Try using alternatively polybrene or protamine sulphate.
5. Try spin infection of cells in suspension.
5. Play around with duration of infection.
6. In my hands Qiagen maxiprep purified DNA gives best efficiency.
7. You can repeat infections may be up to 3 times with atleast 12 hrs interval.
Hope these would help.
Good luck.

[Functional Screens]

You might want to consider the following factors.
For transfection: cell line; DNA quality; transfection methods; transfection reagents plus/minus enhancer such as protamine sulfate, chloroquine, sodium butyrate; Concentarting virus with ultracentrifugation, etc.
For transduction: polybrene, spinoculation, multiple transduction, more concentrated virus with less cells, and etc.

To transduce hematopoëtic stem cells, you might need spinoculation with polybrene.