Development of real-time detection system for ovarian biomodulators using Luciferase reporter gene and its application.

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The reporter gene assay is a useful system on real-time measurement for expression and action times of paracrine and autocrine factors functioned in cell-to-cell, germ cell-to-somatic cell, and embryo-to-somatic cell communications. The system is consisted of the luciferase gene vector (pGL3) constructed with the regulatory domain of target genes, which is transfected into the ovarian cells, such as granulosa cells, and undifferentiated cells, such as embryonic myoblasts. For example, the CRE reporter vector was constructed ; in brief, oligonucleotides containing 3 tandems of consensus CRE (TGACTGCA) were inserted into the Bgl II and Mlu I sites of pGL3-Promoter vector. The CRE-constructed plasmid vector was transfected into granulosa cells isolated from porcine ovaries. The granulosa cells were cultured with porcine oocytes in the presence of luciferin a substrate of luciferase upon FSH stimulation. The presence of oocytes reduced the action of FSH, suggesting that oocytes release inhibitory substance(s) into granulosa cells. Studies on differentiation of myoblasts into myotubes showed the increase of Smad activity with the Smad responsive element. Furthermore, the progesterone receptor element, androgen receptor element and estrogen receptor element were similarly constructed with pGL3 vector, and real-time measurement of the respective promoter activity was developed. However, the system should be further developed for short half-life of luciferase, transfection efficiency of plasmid vector into cells, and inhibition of apoptosis of transfected cells.