retrovirus transfection in NIH3T3 - (Feb/23/2007 )
Hi, guys
I have tried to use retrovirus to transfect NIH3T3 cells since one month ago. So frustrating! The transfection efficiency is too low to be counted. I almost couldn't find out any proliferating cells after 7 days 350ug/ml Zeocin selection. Please don't laugh at me. I know NIH3T3 is the easiest cell line for transfection.
Here is what I did:
two vectors (pMIG and pLXIZ) have been used, insert is a membrane protein with PLAG tag
Lipo2000 is used to transfect package cell (pheonix-Eco, which is 293 modified)
Day 1 I transfected plasmid to package cells in the afternoon
Day 2 I change medium in the morning
Day 4 I collected virus medium in the morning, and use ALL virus medium to infect NIH3T3 with 6ug/ml polybrene
Day 6 I either add Zeocin to select sable transfected cells or lysis cells to check transfection efficiency
I think the problem is there are no virus been produced or too little to be used.
One day after transfection, my eco package cells tend to aggregate together, I didn't see many cells died though even one week after transfection.
Are there any morphology changes in eco package cells during virus production? How can I know there are virus produced?
Will eco cell die after virus production?
Thanks a lot
Yue
You viral titres might be too low. You need to check your titres with a GFP or a control fluorescent vector. Infect your 293 cells also to verify titres.
After transfections, cells do tend to join together, these cells are supposed to be transfected with the plasmids and involved in virus production. The cells become exhausted after a period of viral production.
Are your NIH still dividing? Because retrovirus can only infect dividing cells. You may also loose some virus with the medium change at day 2.
Hope that helps!
And don't worry, laughing at you would be really immature 
Hope that helps!
And don't worry, laughing at you would be really immature
How big is your retroviral vector in total (including the gene/cDNA of your interest)? If close to 10kb, then, the viral packaging efficiency can be very low. In this case, you can still transfect cells but the infection may not work well.