Google Groups no longer supports new Usenet posts or subscriptions. Historical content remains viewable.
Dismiss
Hygromycin-B-Phosphotransferase(HPH) as selectable marker
0 views
Skip to first unread message
Lori E Hutchinson
Feb 24, 1995, 8:02:34 PM2/24/95
to
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.
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
ken...@iastate.edu
mpk6
Feb 27, 1995, 10:57:10 AM2/27/95
to
I had exactly the same problem when using the dominant selection with the relat
ed Tn5 marker which provides resistance to the antibiotic G418, another amino g
lycoside. We selected cells at a final concentration of 800 microgram/ml in med
ium containing 10% fetal bovine serum. It turns out that the problem was defini
tely solved by plating the cells at a very low density prior to selection. For
some reason you obtain many background false positive colonies if the initial c
ell density is too high. Hope this helps.
ed Tn5 marker which provides resistance to the antibiotic G418, another amino g
lycoside. We selected cells at a final concentration of 800 microgram/ml in med
ium containing 10% fetal bovine serum. It turns out that the problem was defini
tely solved by plating the cells at a very low density prior to selection. For
some reason you obtain many background false positive colonies if the initial c
ell density is too high. Hope this helps.
Best regards,
Mike Kladde
PennState Univ.
308 Althouse Lab
University Park, PA 16801
0 new messages