Hygromycin-B-Phosphotransferase(HPH) as selectable marker

Lori E Hutchinson kendall at iastate.edu
Fri Feb 24 20:02:34 EST 1995

We are trying to stably transfect Rat basophilic leukemia (RBL) cells using
HpH as a dominant selectable marker. We are not having any luck killing
untransfected  cells with hygromycin-B. Several sources have suggested a drug
range of between 50-400ug/ml for adequate killing. We have resorted to using 
750ug/m in media containing 15% serum and while the proliferation rate was been
slowed compared with that of cells in media alone, there are still many,
adherent, living cells. There are concentrated foci of cells here and there
that look like they may have originated form a single possibly "resistant" 
clone,but the background of living cells is still high. Everytime I change the
media with media containing fresh drug (every 4 days) the cells seem to do even
better!This has been going on for three weeks. Has anyone out there had similar
trouble killing cells with hygromycin? Could the high serum in the media be 
the problem? What about stability of Hygromycin-B in aqueous stock solution?
Sigma tech rep told me it was stable "forever" in water?
Any help would be appreciated.

Lori Hutchinson 
Lori E Hutchinson
kendall at iastate.edu

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