I'm having trouble packaging lentivirus in HEK293T using pCMV, pMDG, an shRNA and polyfect. I normally seed 1.5 x 106 HEK293T cells in 60 mm on Day 0, which translates to about 60 to 70% confluence on Day 1. Does anyone have long term experience working with 293T and can offer a working protocol? or can point me in the right direction with possible points of failure? Thanks.
we do this pretty routinely in my lab.
two questions:
1. Is this the first time you've packaged lentivirus in 293T cells or have you had success before?
2. how do you know that you have failure to produce lentiviral particles?
3. do you filter your supernatant and if so, what filter do you use?
Troubleshooting tips:
We've found it very helpful to use GFP or another fluorescent protein in a lentiviral backbone as a positive control.
- You should be able to see >75-80% transfection efficiency in your 293T cells based on GFP expression
- you should see syncytia formation by day 3 post-transfection due to VSV-G expression
- your target cells should become GFP+
- Badges
- Science topic