Knock-down og FEN1 in HeLa cels

Abstract

In order to assess the biological role of the flap endonuclease 1 (Fen1), RNA interference (RNAi) was chosen as a method for FEN1 down-regulation in this study. Knock down of FEN1 was achieved both in stable transfected HeLa cells, using vector-based method, and by transient transfection using siRNAs. For transient assays, knock down was monitored by the down regulation of green fluorescence protein (GFP) expression, the GFP gene was fused with the Fen1 gene. In the vector-based method, knock down of FEN1 was monitored by Western analysis.

HeLa cells deficient for FEN1 expression was investigated for phenotypical changes such as growth retardation and sensitivity to DNA damaging agents. Findings indicated that HeLa cells deficient in FEN1 expression displays prolonged cell cycles, spending twice the time compared to wild type HeLa cells. These results indicate a role of FEN1 in replication, and confirm the findings of FEN1 in processing flap structures in Okazaki fragment processing. The FEN1 down-regulated HeLa cells were also investigated for sensitivity to DNA damaging agents. These results indicated the amount of FEN1 was not limiting for recovery from such agents.