Establishment of Cell Line with the FEN-1 Gene Blocked by Its Antisense.

FEN-1 is a structure-specific endo/exonuclease, which is involved in the process of both DNA duplication and DNA repair. In this work a mammalian expression vector expressing antisense FEN-1 gene fragment pMAMneoAmp(-)FNB(-) was constructed, after cloning the NcoI-BamHI fragment of FEN-1 gene into the mammalian expression vector pMAMneoAmp(-) in antisense orientation. After FL cell was transfected with pMAMneoAmp(-)FNB(-) and selected by G418, the FL-FEN-1(-) cell line, in which the FEN-1 gene expression was blocked, was established. It was found that the growth of FL-FEN-1(-) was decreased upon the induction with dexamethasone and its T(D) was 3.03 d, while the T(D) of controls FL and FL-M induced with dexamethasone was 2.03 and 2.22 d, respectively, and the T(D) of the FL-FEN-1(-) cell without dexamethasone was 2.38 d.

The Influence of FEN-1 Gene on Cell Cycle and Genetic Stability.

FEN-1 is essential in the cell replication, repair and in the maintenance of cellular genetic stability. In this report, it was verfied that FEN-1 antisense mRNA fragment was expressed in the cell line FL-FEN-1(-),constructed in our lab, blocking FEN-1 gene expression. It was found by the flow cytometer analysis that the cell cycle of FL-FEN-1(-) cells was delayed in the S-phase DNA synthesis process and arrested in G(1) phase. In a mutation assay, based on the shuttle-plasmid pZ189, the spontaneous mutation frequency of SupF tRNA gene in the plasmid in the FL-FEN-1(-) cells was 19.1x10(4),while it was 2.9x10(4) and 3.0x10(4) in the control cells FL and FL-M, respectively. Further study showed that nontargeted mutation frequency of the FL-FEN-1(-) cell induced by MNNG was almost the same as the control, indicating that the mutants derived from the block of FEN-1 gene and the nontargeted mutants may be formed through different passways. The FL-FEN-1(-) cells exhibit increased sensitivity to alkylating agent MNNG.