gms | German Medical Science

56. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie e. V. (DGNC)
3èmes journées françaises de Neurochirurgie (SFNC)

Deutsche Gesellschaft für Neurochirurgie e. V.
Société Française de Neurochirurgie

07. bis 11.05.2005, Strasbourg

High efficiency transfection of glioblastoma cell-lines and primary cells using electroporation by nucleofector technology

Hoch-effiziente Transfektion von Glioblastom-Zelllinien und Primärzellen mit Hilfe von Elektroporation durch Nuclefector-Technik

Meeting Abstract

  • corresponding author J. Stojic - Neurochirurgische Universitätsklinik, Tumorbiologisches Labor, Würzburg
  • C. Hagemann - Neurochirurgische Universitätsklinik, Tumorbiologisches Labor, Würzburg
  • C. Meyer - Neurochirurgische Universitätsklinik, Tumorbiologisches Labor, Würzburg
  • S. Gerngras - Neurochirurgische Universitätsklinik, Tumorbiologisches Labor, Würzburg
  • G. H. Vince - Neurochirurgische Universitätsklinik, Tumorbiologisches Labor, Würzburg
  • K. Roosen - Neurochirurgische Universitätsklinik, Tumorbiologisches Labor, Würzburg

Deutsche Gesellschaft für Neurochirurgie. Société Française de Neurochirurgie. 56. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie e.V. (DGNC), 3èmes journées françaises de Neurochirurgie (SFNC). Strasbourg, 07.-11.05.2005. Düsseldorf, Köln: German Medical Science; 2005. DocP183

Die elektronische Version dieses Artikels ist vollständig und ist verfügbar unter: http://www.egms.de/de/meetings/dgnc2005/05dgnc0451.shtml

Veröffentlicht: 4. Mai 2005

© 2005 Stojic et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

Objective

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.

Methods

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.

Results

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.

Conclusions

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.