I want to stably transfect MCF-7 cells with caspase-3 GFP plasmid. I've not performed stable transfections in the past. So, I'm looking for a protocol which will help me to do that.
Stable transfection is (trivially) more difficult than transient transfection. We've pre-optimized a transfection reagent specifically for MCF-7 cells (https://altogen.com/product/mcf-7-transfection-reagent-breast-cancer-htb-22/) and we've done several successful stable transfections with it. You should aim to have a good selection marker, and expect around 20% efficiency for stably-transfected cells (which is established after several generations have grown).
Hi, simply add a selection marker to your plasmid. Most used method is to take a resistance to an antibiotics. Good luck!
Hi Punita,
I hope that this document will be useful. Good Luck.
https://worldwide.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/transfection/
Hi,
For stable pool generation put your transfected cells under selection pressure ( usually MTX -DHFR based or MSX - GS System)and antibiotic selection marker based on your vector starting from lower concentration to higher selection range. Selection range is established based on a kill curve experiment initially or based on the established experimental evidence available.Once you have stable selection pool you can go for limiting dilution cloning ( single cell isolation) for obtaining homogeneous selected population.
Good luck!
Hi
I would suggest using a retroviral based expression system preferably with puromycin selection. Also one where HEK293 can be used as packaging cell line. For selection prepare a kill curve first and select the dose as described previously.
Prior to transfection, determine the selective drug concentration required to kill untransfected cells (kill curve; Ausubel et al. 1995).
Stable transfection is (trivially) more difficult than transient transfection. We've pre-optimized a transfection reagent specifically for MCF-7 cells (https://altogen.com/product/mcf-7-transfection-reagent-breast-cancer-htb-22/) and we've done several successful stable transfections with it. You should aim to have a good selection marker, and expect around 20% efficiency for stably-transfected cells (which is established after several generations have grown).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line