Dear Salma Bedaiwi

It happened with me too that after DNA transfections and selection by antibiotic, the efficiency was literally negligible.

I would advise you to go for a lentiviral system for generating stable cell lines of your requirements. Since there are many unknowns, it could be any number of reasons ranging from antibiotic concentration or even the function of the transgene. But in my experience, viral systems are better for downstream selection of single mammalian cell clones. Best of Luck.

Our lab uses VSV-G-pseudotyped lentivirus system which combines 3 plasmids DNA ( packaging, envelope and target DNA) this 3 DNA will mix in a proportion of 0.5 : 0.5 : 9. Then follow the Lipo. 2000 transfection protocol provided by ThermoFisher, 2.5~4ug of total DNA in Opti-MEM into Lipo. 2000 in Opti-MEM with same volume, after 20 mins DNA-Lipo. mixture will directly into a single well containing ~1x106 seeded cells (293) in 6 well plate. Collect medium for 3 days, then condense (or not) this sup. to obtain a higher titer of virus, the virus sup. will use to infect target cells. In your case, if the selected cell do express GFP, than I'll suggest you to sequence your DNA, to make sure if zeocin resist. sequence is in frame, otherwise you can use a negative and positive control to compare with your target DNA transfected MCF-7. The one last thing is, have you test your cells with the appropriate concentration of zeocin (a concentration gradient test maybe?), which different cells may differ in resistance and toleration.

Hi Mu-Shen Chang and Aadil Javed,

Thank you for your response. For the lentivirus system, I am wondering about the safety. Would I need special safety precautions?

"The one last thing is, have you test your cells with the appropriate concentration of zeocin (a concentration gradient test maybe?)" Mu-Shen Chang

I did the kill curve. I couldn't go higher than 250 mg/ml because of the toxicity of the lipofictamin. However, I found some papers that recommend 500 mg/ml for MCF-7.