Stable Transfection of large DNAinto 293 cells - (Aug/08/2001 )

Hi, I tried several times to transfect a 9kb and 16kb cDNA inserted in pEGFP-N1 vectorto establish a stable cell line (HEH293), but I failed even cut the circle DNAinto linear DNA. Some cell colonies could survive in G418 medium, but no fluroscence under microscope and I could not detect the expressed protein by Western-Blot. For transient transfection, the expression was very well. Doesanyone have good suggestion about stable transfection with large DNA?

Thanks in advance.

Zhendong Zhao

Hi,I also transfected a cDNA inserted in GFP expression vector to fibroblast .It was sucessful.I think you can try to determine the optimal drug concentration and plating density.For stable transfection,it is good to seed low density.For vary batch cells and drug,the datas maybe different.But for some cell lines ,the green fluorescence will be fade away after some times.I had a stable transfected cell line with gfp plasmid,at first the cells were green,but after one month the green color was weaker than before.