知识与财富的链接
目的 探究Nanog启动子-332/+50区与双荧光素酶报告载体pGL4.10连接后负向调节Nanog-135/-230区活性的调节序列。方法 截短法将Nanog启动子-812-+171区不同片段与pGL4.10载体连接,脂质体转染法将各载体分别与内参载体pRL-TK共转入细胞,双荧光素酶系统报告系统检测报告基因的表达变化。结果 Nanog启动子-812/-332区、+50/+171区及-332/+51(或+56)区均不含Nanog-135/-230区的负向调控元件,改变Naong-332/+50区与报告载体pGL4.10的连接位点(NheⅠ,BglⅡ)行成序列“-GCT(A)GCGAGATC-”即可消除Nanog启动子-332/+50对报告基因的正向调控作用。结论 待检测启动子与载体连接构成“-GCT(A)GCGAGATC-”序列后可负向调控Nanog-135/-230区的活性。
Objective Identify the regulatory sequence of Nanog promoter -332/+50 that down-regulates Nanog -135/-230's activity. Methods Construct a set of plasmids that ligate different Nanog promoter -812-/+171 truncates with pGL4.10, co-transfect pRL-TK with the plasmid minded to cells using Lipofectamine 2000, testing the promoter activity using the Dual Luciferase Reporter assay system. Results There have no regulating sequence in the Nanog promoter -812/-332, +50/+171, and -332/+51. A change of ligated sequence between the NheⅠ, BglⅡ restriction sites maintained in pGL4.10 and Nanog promoter -332/+50 sequence, “-GCT(A)GCGAGATC-”, could counteract the regulatory effect of Nanog promoter -332/+50 on report gene expression. Conclusion Sequence of “-GCT(A)GCGAGATC-” could reversely regulate Nanog 332/+50 promoter activity.