I am trying to generate a stable clone in HCT cell line using G418. What I have found is that although I am using concentration gradient ranging from 200ug to 2400ug / ml. I am seeing cell quite normally growing well attached and increasing in number, as if I putting supplement to them. I tried to do the kill curve with different passage but failed. Now what I want, if anyone of you have experience with HCT stable clone using G418, please tell what concentration I should use and for how many days I need to keep the cells for selection.
Will remain thankful for this help.
Well, the range of concentrations that you use is broad enough to kill about any cell line. I can see two possibilities here: 1) your antibiotic is not potent/expired. You can test it's potency on a garden variety cell line using the same range of concentrations; 2) your cell line is already G418 resistant (perhaps, made so in the process of being made -/- for unspecified allele). This can be tested by PCR. Just make sure to test two sets of primers: for Tn5 and Tn903 nptii
I have tested the same G418 on other cell lines and its working fine. But I have found publication where they have kept cells in dish for one month in G418 containing media. I have started the selection........ Lets hope for the good
Can you rule our that your cells are already G418 resistant? As your question suggests, HCT116 are not WT, but rather -/-. That suggests that it went through two rounds of transfection and selection (to inactivate each allele). Often, this involves introduction of an antibiotic-resistance marker.
True, this is a knockout cell line. Human adenocarcinoma, if I am not mistaken. One of the very few, in which homologous recombination is efficient enough to do gene knockout by homologous recombination. Gene targeting typically involves transfection of a cell line with targeting construct, selection of transfected clones using antibiotic, screening for the clones, in which integration occurred by homologous recombination in one allele, and repeating the procedure with another allele. If G418 resistance marker was used for targeting and not removed later, your cells may be resistant to G418
That I need to check in that case. But people have produced stable clones in HCT 116 knockout by using G418. Still I need to check on that. But your point is valid enough to check
For the general info, HCT116-/- cells are inherently resistant to hygromycin and neomycin as knockout selection is done using these antibiotics. Yes one can go for blasticidin or puromycin, i did with blasticidin.
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