I am trying to stably express either a WT or a mutant form of my protein of interest under the control of doxycycline (using pTRIPZ vector) in C2C12 and ST2 cells to study it's role in bone development and remodeling. However, I am having troubles with infection efficiency. I have tried the same virus on 293T cells and it works nicely, so quite sure that it is the efficiency problem. If anyone has any ideas or suggestions to improve the same, it will be highly appreciated. Thanks in advance.
You might try different MOIs or check the confluency of the cells, sometimes when cells are confluent are poorly transduced. This link has all of the possible techniques that will help you to establish a cell line using lentiviral vectors: https://www.thermofisher.com/qa/en/home/references/gibco-cell-culture-basics/transfection-basics/factors-influencing-transfection-efficiency.html
Two major possibilities here:
1. Your target cells are not easy to infect. 293T cells are very permissive to lentivirus, I notice that both your target lines are from mice, mouse cells are often resistant to LV infection.
2. Your transgene promoter works fine in human (293) cells but not in mouse cells.
The way to tell is to do a PCR-based VCN assay on your target cells. If you can identify integrated virus then you know that the LV infects your cells but nothing expresses. If you find no evidence of integration even at a high MOI, then your target cells may be difficult to infect
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line