一种适于家蚕细胞激素研究的双荧光素酶报告基因系统的内参质粒的构建

Establishment of a suitable control reporter plasmid of a dual luciferase reporter gene system for hormone research in silkworm cell lines

摘要:

双荧光素酶报告基因系统能够提供灵敏的读数，但该系统需要依赖组成型表达的内参对读数进行归一化。然而，大多数内参并不是在所有条件下都组成型表达。为此，文中建立了一个有效的方法制备适于家蚕细胞双荧光素酶报告基因系统的内参质粒。首先，突变BmVgP78启动子上的激素应答相关元件，获得了在家蚕细胞中稳定表达的组成型启动子BmVgP78M；然后，用BmVgP78M替换pRL-SV40质粒上的SV40启动子和嵌合内含子序列，成功构建了pRL-VgP78M内参质粒；最后，通过细胞转染实验证实pRL-VgP78M内参在家蚕细胞系中稳定表达，并且pRL-VgP78M内参的表达活性不受蜕皮激素、保幼激素及激素相关转录因子的影响。最终，获得了在家蚕细胞中稳定表达且表达量适中的内参质粒pRL-VgP78M。该内参可以有效地作为双荧光素酶报告基因系统的内参质粒用于家蚕细胞系中激素的研究。同时，该内参质粒的构建方法也为构建适于其他物种细胞系的双荧光素酶报告基因系统的内参质粒提供了参考。

Abstract:

The dual luciferase reporter gene system provides sensitive readout, while it relies on a constitutively-expressed control gene for readout normalization. However, most standard control reporter genes are not constitutively expressed under all conditions. Here, we report an effective method to construct a control reporter plasmid for the dual luciferase reporter gene system that would be suitable for hormone research in silkworm cell lines. First, we modified BmVgP78M, a stably-expressed constitutive promoter in silkworm cells by mutating its hormone-related element. Then, we constructed the pRL-VgP78M control reporter plasmid by replacing the SV40 promoter and chimeric intron sequences in pRL-SV40 with the BmVgP78M sequence. Finally, we confirmed that the pRL-VgP78M control reporter plasmid could be stably expressed in silkworm cell lines via cell transfection experiments, and it was unresponsive to the induction of ecdysone, juvenile hormone, or their transcription factors. We thus obtained a control reporter plasmid pRL-VgP78M that could be expressed stably and moderately in silkworm cells. It can be readily used as the control reporter plasmid of the dual luciferase reporter gene system for hormone research in silkworm cell lines. It will also provide a reference for construction of control reporter plasmids of dual luciferase reporter gene systems that are adaptable to cell lines isolated from other species.