Construction and Application of RARE-regulated Luciferase Reporter Gene System Stimulated by Retinoid Acid

doi:10.13560/j.cnki.biotech.bull.1985.2015.06.033

Abstract

Abstract: Biostearin plays a crucial role in the development of metabolic diseases by target gene regulation of retinoic acid(RA)in metabolically active tissues and interaction with circadian pathway. The luciferase reporter gene system regulated by retinoic acid response element(RARE)was constructed and expressed in 293T and primary hepatocyte of a mouse, which provides the possibility for studying biostearin and circadian, as well as signaling molecules of upstream regulation of target genes in researching metabolism. By PCR-based site-specific mutagenesis, RARE promoter was inserted into multiple clone sites of pGL3-Basic vector. In 293T cells, we detected the gene expression and half maximal effective concentration(EC50)of RARE responding to RA and screened the possible target genes of regulating RARE. The pGL3-RARE-Luc plasmid and pShuttle vector were digested by restriction enzyme and ligated. Through transformation into competent cell BJ518 the recombinant adenovirus vector Ad-Basic-RARE-Luc was obtained and amplified after transfection to MGH cells. The activity of recombinant adenovirus was detected in primary hepatocyte of a mouse with Dual-luciferase Reporter Assay System. The results indicated that luciferase reporter gene vector regulated by RARE in 293T cells was stimulated by RA. RARβ promoted the stimulation of RA in RARE regulation, and CRY1 prohibited the response of RARE-Luc to RA. The adenovirus vector Adeasy-Basic-RARE-Luc responding to RA stimulation in primary hepatocyte of a mouse was constructed successfully.

Key words: retinoid acid, RARE, luciferase reporter gene, circadian, metabolism

Yuan Yuan, Chen Yaqiong. Construction and Application of RARE-regulated Luciferase Reporter Gene System Stimulated by Retinoid Acid[J]. Biotechnology Bulletin, 2015, 31(6): 209-215.

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