I tried to stably transfect Hek293T with linearized pEF6/V5 plasmid, that contain the blasticidin resistance gene. The negative control cells (no-transfected Hek293T) died into about one week. I plate the transfected cells at 25% confluence (recomendation of datasheet) and add 10ug/ml of blasticidin. Three days after transfection, I saw about 9% os positive cells by FACS. I kept the cells in selection medium. The cells grew slowly, I changed the medium each 3 days, and after 10 days I've checked it by flow cytometry and I got only 0,7% of positive cells.
Can anyone help me, please? Did someone get a stable cell line with blasticidin?
I hate to say this, but HEK293T cells is not good for stable transformation especially when you use vectors carrying SV40 ori, which pEF6/V5 has. It is because HEK293T cells are expressing large T antigen, which promote replication of DNA having SV40ori. When plasmids having SV40ori is integrated into chromosomes, replication of the chromosomes would not be regulated properly, resulting in an increase in chromosomal abnormality. This means that the cells could change easily. I think you can find such descriptions from invitrogen because they are selling 293T or subclones of 293T. (it is controversial, but I know some actually get stable transformants by using plasmids carrying SV40ori in 293T)
Another possible reason for the decrease in the expression level could be losing episomal plasmids in cells because plasmids having SV40ori can replicate by themselves in the presence of large T antigen.
It may be possible that you were able to pick up single clones expressing it well. But it is already 7 out of 1000... You might want to pick up single clones at initial selection.
I hate to say this, but HEK293T cells is not good for stable transformation especially when you use vectors carrying SV40 ori, which pEF6/V5 has. It is because HEK293T cells are expressing large T antigen, which promote replication of DNA having SV40ori. When plasmids having SV40ori is integrated into chromosomes, replication of the chromosomes would not be regulated properly, resulting in an increase in chromosomal abnormality. This means that the cells could change easily. I think you can find such descriptions from invitrogen because they are selling 293T or subclones of 293T. (it is controversial, but I know some actually get stable transformants by using plasmids carrying SV40ori in 293T)
Another possible reason for the decrease in the expression level could be losing episomal plasmids in cells because plasmids having SV40ori can replicate by themselves in the presence of large T antigen.
It may be possible that you were able to pick up single clones expressing it well. But it is already 7 out of 1000... You might want to pick up single clones at initial selection.
you could try to increase the DNA quantity during transfection in order to transfect more plasmid copies per cell, increasing the possibility of integration...
- Badges
- Science method