Submitted by Casvanas t3_104dssn in askscience
I believe I understand the basics of the process.
You have a gene you want 'disabled', you engineer a specific DNA sequence that contains the neomycin-resistance gene from bacteria as a marker and pray it gets successfully incorporated into the genome of an embryonic stem cell (ESC) and 'knocks out' whatever gene you want through homologous recombination.
Afterwards you let the populations of ESCs grow a bit in culture. After a while, your petri dish contains some populations in which the process was successful and a whole lot in which it wasn't. That's where the neomycin-resistance gene comes into play; you add neomycin to the petri dish and soon it becomes obvious in which cells the homologous recombination was successful and in which it wasn't as the cells that didn't get the neomycin resistance gene die off. At least that's the way I understand it.
However, how does neomycin kill these (eukaryotic) mouse cells? The way I understand it, neomycin should only be able to 'kill' bacterial cells as they specifically inhibit bacterial protein synthesis. What am I missing?
Lepmuru t1_j36hlia wrote
You are 100% accurate with your assessment. The only thing keeping you from understanding is an assumption you made involuntarily (which also is kind of non-trivial).
You select for neomycin-resistance, but not by adding neomycin. Instead, to select eukaryotic cells you would use a substance called geneticin or G418 which is toxic to eukaryotic cells, but cleared by the neomycin-resistance gene.