Luciferase Stable Cell Line

Establishment of an Evaluation Method for siRNA Delivery by Using HeLa-luc Cells or CHO-luc Cells Carrying the Luciferase Reporter Gene(C Lai, et al.,2006)

We should use inducers to express luciferase in these HeLa-luc cells and CHO-luc cells. We considered that it is better to choose a shorter incubation time with inducers and lower inducer concentrations, because the inducers were shown to be cytotoxic for these mammalian cells. And, we considered that the number of seeded cells should be set to achieved was 90—95% confluent in luciferase assay. Thus, we optimized the inducing conditions for HeLa-luc cells and CHO-luc cells accordingly. In the case of HeLa-luc cells, when the number of seeded cells was 6103 cells/well, about 95% confluent was achieved in luciferase assay. For the inducer concentrations of 5 ng/ml of PMA and 0.25 mM of A23187, luciferase was expressed at low level. However, when the inducer concentrations were 10 ng/ml of PMA and 0.5 mM of A23187, luciferase was expressed at high level. Luciferase was expressed at high level in 6 h incubations with inducers, which was the shortest time condition of all (all of the conditions are indicated by the asterisk in Fig. 4A). In addition, when the HeLa-luc cells were incubated for 24 h with inducers, their shapes were changed. In the case of CHO-luc cells, when the number of seeded cells was 8103 cells/well, about 95% confluent was achieved in luciferase assay. For the inducer concentration of 1 mM of Dex, which was the lowest concentration of all, luciferase was expressed at high level. The CHO-luc cells were less sensitive than HeLa-luc cells, so we considered that favorable incubation time was 7 h, in which the highest luciferase activity in the condition of 8103 cells/well for the number of seeded cells and 1 mM of Dex (all of the conditions are indicated by the asterisk in Fig. 4B). Furthermore, in the case of HeLa-luc cells, cellular cytotoxicity appeared after 24 h of incubation with more than 0.4 ml of Lipofectamine2000/well. At incubations of 48 h, however, cellular cytotoxicity appeared with more than 0.05 ml Lipofectamine2000/well (Fig. 5). It was considered that total incubation time with Lipofectamine2000 had to be 24 h for HeLa-luc cells. We outlined the optimised experimental conditions for siRNA transfection in HeLa-luc cells and CHO-luc cells in Chart 2.