gms | German Medical Science

Glioblastoma cells, especially primary cells, are difficult to transfect. High transfection rates can only be achieved by viral infection, which may alter the cell's phenotype. By usage of a liposome-based transfection method Zerrouqi et al. obtained 36% transfection efficiency, which is too low for many cell-based assays. Therefore, we decided to test electroporation by nucleofector technology (Amaxa), which promises high efficiency transfection with no alteration of the cell's phenotype for transfection of different glioblastoma cell lines and primary cells.

We optimized the nucleofection protocol (Amaxa) for high efficiency transfection of human glioblastoma cell lines U251, GaMg, U373 and primary cells according to the manufacturers recommendations using a GFP expressing plasmid. We then examined the phenotype of transfected cells in comparison to untreated cells in proliferation-, migration- and MTT-assays, checked whether transfected cells were able to form spheroids and investigated the expression of the glial marker protein GFAP by immuno-histochemistry.

Using these methods we obtained 60-90% transfection efficiency of different glioblastoma cell lines and primary cells using solution V and transfection program T20. The transfected cells expressed GFP without further selection up to seven days, and retained their ability to form spheroids, a characteristic, which is quickly lost if there are phenotypic alterations. However, we detected a slight change in the phenotype, since transfected cells showed a short delay in proliferation and spheroids formed slower and remained smaller compared to untreated cells.

Electroporation by nucleofection technology is suitable for high efficiency transfection for overexpression or RNAi studies. The transfection protocol optimized for the U251 glioblastoma cell line can also be used for transfection of other glioblastoma cell lines and even primary cells. However, compared to untreated cells there is a delay in cell proliferation which may be due to a recovery period of the cells after transfection. This transfection method is a highly recommendable tool for functional assays.