lentiviral transdcution of hematopoŽtic stem cells
Posted 14 June 2009 - 05:08 AM
I'm trying to transduce hematopoŽtic stem cells with a lentivirus. Producing the lentivirus is OK. But the transduction is not efficiŽnt enough.
Is there any possibility to make a more efficiŽnt virus? Or to make the transduction better?
Posted 14 June 2009 - 06:00 AM
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.
Posted 15 June 2009 - 07:29 AM
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 hematopoŽtic stem cells, you might need spinoculation with polybrene.