G418-selected cells do not divide - (Sep/12/2006 )

We transfected MDCK cells wit LF 2000 and cultured for 2 weeks in G418; we tried various G418 concentrations between 500 and 1000 µg/ml; we found some stably transfected candidates as single cells and tried to re-culture them in medium without G418 but they do not divide over another 2 weeks; perhaps they did also not divide during selection; has someone similar experiences? Can G418 selection favor a cell cycle arrest? How to release the brake?

maybe it's the gene transfected that inhibit the cell growth?

thanks for reply and the good idea; it´s a receptor kinase were we transfected in controls two different inactive forms; the gene with the functional kinase domain was successfully transfected in other epithelial cells and did not block cell cycle

is there a possibility of a dominant negative effect?

same idea that Fred.
if it's a receptor involved in cell growth (like growth factor receptor) and you overexpress an inactive form, you can inhibit it.
I had the same problem with FRNK , a FAK isoform lacking the kinase domain. the overexpression of FRNK inhibits FAK, and consequently the cell growth. I had to do transient transfection.

thanks, all good ideas; what I have to add is that our mock containing kanamycin/neomycin resistance gene also refuses to divide; is there a phase lag where selected cells have to recover from G418 pressure? and after that start to divide again? of course, as suggested, dominant-negative or inactivating effects may block normal cell division but I mean what is beside these effects and is there a special effect of G418 or the kanamycin/neomycin gene expression on proliferation?

Are doses you are using pretested, the lowest possible dosage that kill most normal cells?

Some cell types do not grow well if cell density is too low. I wonder if this would help: you culture them with conditioned medium. You plate normal cells, grow them overnight, harvest culture medium, filtered it w/0.22 um filter, then add antibiotics for selection and use it instead of regular medium+FBS.

thanks genehunter-1, we try all cells that survive between 500 to 1000 µg/ml where all untransfected cells have died; we have now 10 plates of 96-wells, many candidates, only a very very few divide; tomorrow we will try conditioned medium

hello genehunter-1, we´ve done as you have said to culture with competent media (plus we gave some extra EGF); we now observe growing lamellopodia and better division, so I think we are on the right way, thanks again for your good advice