Hi, I have transfected my HEK293 cells with pcDNA3.1_plasmid of interest with EGFP by using lipotransfection method. Post transfection 48h the egfp expression looks good with more than 50% transfection rate. Started G418 selection (DMEM+10% FBS+ 2mg/mL G418). Post transfection 48h, the cells transfer from 24wp to 12wp to create space for G418 medium to get to non-transfected cells. Medium replacement done daily, and after a week, fresh selective medium is prepared. Massive cell death observed after 2 days of G418 selection.
Day 7, 95% of non transfected cell die. Colonies/ egfp rounded cell clump together can be observed and a lot of resistant cells survived but did not attached under the G418 selective stress.
Day 8, the selective medium reduce to 1mg/mL and Day 10 reduce to 500ug/mL and day 12 reduce to 250ug/mL hoping that the resistant cells will attach and starting to expand.
Unfortunately, the non transfected cells starting to grow rapidly and the cells with EGFP did not grow so much. Any comments and thoughts are welcome. The reason I use 2mg/mL from to start becuase I have done 1mg/mL to kill the HEK293 but it wasn’t effective. The active G418 percentage is 80.9% and I have reconstituted G418 with 100mM HEPES buffer And filtered.
I have read about for the survival cells to expand it might take a long time under the selective pressure. When is the right time to reduce the antibiotic?
the attached image is Day 10 under G418 selection. Should I be more patient and keep the G418 concentration high and wait for the colonies to attach and expand?
My suggestion is to conduct two rounds of selection with the same G418 concentration. Start the first round of selection 24-48 hours after transfection, culture the cells for 2-3(5) days with G418, depending on the death rate of the cells; then let them recover in medium without G418 until you have a reasonable number of live cells, repeat the selection process.
Or make constructs with another resistance cassette e.g. puromycin.
Sabine Strehl thank you for answering! Under G418 selection pressure, the resistant cells did not die but not exert their typical morphology even after 10 days. The non resistant cells die 99% but not completely, as I reduce the antibiotic level the non resistant cells overgrowth than the resistant cells that express EGFP and slowly my resistant cells reduce and die off and non resistant cells overgrowth. I use medium with G418 for 7 days then prepare a fresh one, but on 7th day of the medium, the antibiotic seems wear off the potency after a few water bath thaw.
I will repeat the transfection and selection process. Thank you for your suggestion.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning