Retroviral transduction of T cells
Posted 30 November 2010 - 09:22 PM
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??
Posted 01 December 2010 - 12:57 AM
Posted 02 December 2010 - 03:44 AM
Yes I did activate the T cells. Maybe you are right when you say you grow the virus in 10cm dishes, I used only a small 6 well plate at different concentration of the fugene: DNA. That would definitely be worth a try.
Also could you please tell me what concentration of DNA do you take for transfecting the feeder/ packaging cells.
Posted 02 December 2010 - 04:03 AM
Edited by Rsm, 02 December 2010 - 04:04 AM.
Posted 07 December 2010 - 10:32 AM
Now I am lost....
I plated my feeder cells (Plat E) at 2million cells in 10cm petridish (10ml), in the morning in IMDM +10% FCS (no antibiotics). I transfected them in the evening. I added 3:1 ratio of fugene(microlitre): DNA(microgram)
reaction mixture as follows
DNA - 3microlitre (15microgram)
Fugene - 45microlitre
Serum free media - 542microlitre
Total vol - 600microlitre
I do not see any change in the morphology of cells, as of today (they are still all round and clumped up) Are they dead??
I have replaced the medium after 24hours with medium with selection
What have I done wrong
Could someone please tell me what seeding volumes for a 10cm plates
Also I have looked through so many protocols [ which only confuse me ] After transfecting the cells, do you change the media for the selection media? If yes after how many hours?
Any help advise will be deeply appreciated!!!