title: [Construction and identification of luciferase reporter gene containing mouse T-bet promoter].
abstract: The aim of this study is to clone the mouse T-bet promoter and enhancer, construct and identify the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS for T-bet transcription regulation study and its function in signaling of multiple sclerosis. The promoter and CNS of T-bet were predicted by bioinformatics assay. The predicted fragment of mouse T-bet promoter plus CNS was amplified by PCR and cloned into pGL4.10. The recombinant plasmid pGL4.10-TBX21pr-CNS was transferred into Escherichia coli DH5α. The positive clone was identified by double digestion with Kpn I and Sfi I and DNA sequencing. Finally, pGL4.10-TBX21pr-CNS was cotransfected with pRL-TK into 293T cells and Jurkat cells, pRL-TK and pGL4.10 as a control. The luciferase activity in 293T cells (P = 0.012 2) and Jurkat cells (P = 0.002 2) was higher than that of the control group. A fragment of 1 028 bp mouse T-bet promoter plus 1 308 bp CNS was successfully cloned and the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS was constructed. In 293T cells and Jurkat cells, pGL4.10-TBX21pr-CNS has the promoter functions. This work offers a basic material for the research of T-bet transcription.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Xu
authors.forename: Shoubiao
authors.initials: S
authors.lastname: Zhou
mesh_terms: D000818:Animals; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D008156:Luciferases; D051379:Mice; D009103:Multiple Sclerosis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D020825:T-Box Domain Proteins / Q000235:genetics*; D000098815:T-bet Transcription Factor
chemical_list: D020825:T-Box Domain Proteins; D000098815:T-bet Transcription Factor; D008156:Luciferases