Hi everyone,

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

Retrovirus infects only activated (cycling) T cells, do you activate your cells? You could also try to concentrate your viral concentration by ultracentrifugation. I usually use three 10cm dishes of feeder cells (7ml medium each) per virus, harvest after 48 and 72hrs, and put all supernatant into one 50ml ultracentrifuge tube. Spin 50,000g for 2hrs, resuspend in 7ml medium, and put on the cells. Works well...

rsm

Retrovirus infects only activated (cycling) T cells, do you activate your cells? You could also try to concentrate your viral concentration by ultracentrifugation. I usually use three 10cm dishes of feeder cells (7ml medium each) per virus, harvest after 48 and 72hrs, and put all supernatant into one 50ml ultracentrifuge tube. Spin 50,000g for 2hrs, resuspend in 7ml medium, and put on the cells. Works well...

rsm

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.


many thanks

That depends... I usually use 5ug of each plasmid per 10cm plate. Some people say more, some say less... I guess it would work with 1ug of each as well. You should test it yourself, where you get the best transfection efficiency.

rsm

That depends... I usually use 5ug of each plasmid per 10cm plate. Some people say more, some say less... I guess it would work with 1ug of each as well. You should test it yourself, where you get the best transfection efficiency.

rsm

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!!!

Shiny