Hi, 

The first step is to performe a dose response to know which zeocin concentration is lethal  for your cells.

I work with a concentration of 300μg / mL and the untransformed cells die after 48 hours of contact.

Thanks a lot Aurore!! By any chance do you have any idea if the dosage will vary if we are going to use it on normal HMEC cells?

Please, take into consideration that bleomycin can generate hydroxyl radicals, oxidize lipids, hydrolyze amide bonds of proteins, and initiate both single-strand and double-strand RNA and DNA cleavage. The extent of the DNA cleavage was shown to dependent on the bleomycin concentration and time of incubation (reviewed in [58]). Comparing the potency of four antibiotics (hygromycin B, neomycin, puromycin, and zeocin) in establishing HT1080 and HEK293 cell lines with stable expression of GFP as a reporter, zeocin was demonstrated to be the best selection agent for cell line development (selected populations with the higher GFP fluorescence levels and less false positives), followed by hygromycin and then puromycin and neomycin [59]. However, it was revealed that zeocin was not completely detoxified and still able to cleave DNA in recombinant SK-OV-3 clones despite the stable expression of the Sh ble (Streptoalloteichus hindustanus bleomycin resistance gene, encoding the Sh protein with a strong binding affinity for the phleomycin family of antibiotics), which confers resistance to zeocin [60]. Authors warned that the cumulative mutagenic damage and adaptive response resulting from the chronic exposure to zeocin for a large number of divisions can have the significant biological consequences, and one should be very cautious in the interpretation of data involving stable cell lines selected with zeocin.

Please see the following manuscript for details:

Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.

Also, I recommend this manuscript

Stepanenko AA, Dmitrenko VV HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene. 2015 Sep 15;569(2):182-90. doi: 10.1016/j.gene.2015.05.065.