Problem in Stable transfection - (Aug/22/2011 )
I was transfected the pBlast42-mcs into CHO cell to express my gene and using 10ug/ml of Blasticidin to kill the untransfected cell. After 14 days, I lysis the CHO cell and run PCR, but there have no desired gene inside the cell. The unknown problem is my CHO is surviving in blasticidin without containing the pBlast vector. My senior also face the same problem using blasticidin and zeocin antibiotic. Anyone can help?
Have you tested your untransfected cells with the blastocidin to see what concentration you need to kill them?
Thank you for reply.
yes, last time i do the optimisation in 2, 4, 6,8,and 10ug/ml and in 6 ug/ml and above,all cell was die. so in frist time, i select in 6ug/ml. For 2nd time transfection I increase to 10 ug/ml for selection, after14 days, I subculture to 2 flask, one is 10ug/ml other is 50ug/ml, but the cell is still survive. Can I try to other cell line like HEK293, may be the CHO cell is gaint the resistance gene?or the pBlast is not in CHO cell?
For my Blasticidin, it is light-sensitive and lasts half a year at -20C. For my Zeocin, it lasts about one year. At 4C, they may last a week or two.