I did a retroviral tranduction of T cells (isolated from mice spleen) and have failed to transduce the cells. I am quite new to the technique and its not been done previously in my lab. Could someone please advise.
Briefly I am pMSCV as my retroviral vector and Plat E cells as my packaging cells. I grew the packaging cells at about 80% confluency (2million cells in 6 well plates) and used Fugene 6 (reaction mixture 3:1, 3:2 and 6:1 of Fugene (μl):DNA (μg), changed the medium after 24 hrs with antibiotics. Harvested the virus after 3 days.
Infected the T cells (5million cells in 6 well plates in complete medium, IMDM) with 1 ml of virus, centrifuged for 90mins at 2000rpm. Next day added another 1ml of the virus centrifuged for 90 mins at 2000rpm. Added fresh media on day 3
Followed by FACS on day 4 (I have a YFP protein tagged to my gene of insert and the retroviral vector contains a HuCD2 gene) and not seeing any difference between the untransduced and transduced cells..

Could someone please advise as to what went wrong and how can I get it to work??
Please help
Shiny