Hygromycin-dependent selection of stable transfectants - (Nov/21/2007 )
against which resistance factor I select with hygromycin? is it ampicillin resistance? what are good concentrations to start with?
The first question I am not sure I understand. If you are talking about bacteria selection, I think it should depend on the vector, not hygromycin.
As for the concentration: According to the producer, the range is 10-1000ug/ml. But when we start the curve, we use 100-1000ug/ml. We found most cells die at about 400-500ug/ml. Some, like Hela, dies at 200ug/ml. This is after 3-4 days, if I remember correctly.
The first question I am not sure I understand. If you are talking about bacteria selection, I think it should depend on the vector, not hygromycin.
As for the concentration: According to the producer, the range is 10-1000ug/ml. But when we start the curve, we use 100-1000ug/ml. We found most cells die at about 400-500ug/ml. Some, like Hela, dies at 200ug/ml. This is after 3-4 days, if I remember correctly.
some use hygromycin to select stable (eucaryotic) transfectants; which resistance factor was(co-)transfected?
I used it on 293. 400 ug/ml hygromycin for 4 days. The resistance gene was Hyg (abbreviation in the vector). The vector was from Clontech. I forgot what mechanism it was for the resistance phenotype, but probably it is a transferase.
http://www.hygromycin.com/
The resistance gene codes for a kinase (Hygromycin phosphotransferase, HPT) that inactivates Hygromycin B through phosphorylation.
http://www.hygromycin.net/
Resistance to hygromycin is conferred by hygromycin B phosphotransferase (Hph). Hph catalyzes the phosphorylation of the 4-hydroxyl group on the cyclitol ring (hyosamine), thereby producing 7"-O-phosphoryl-hygromycin B which totally lacks biological activity both in vivo and in vitro