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

报告基因检测是一种有用的系统，用于实时测量细胞间、生殖细胞与体细胞间以及胚胎与体细胞间通讯中分泌和自分泌因子的表达和作用时间。该系统由以靶基因调控域构建的荧光素酶基因载体（pGL3）组成，将其转染到卵巢细胞如颗粒细胞和未分化细胞如胚胎成肌细胞中。例如，构建CRE报告载体；简言之，将含有3对共识CRE （TGACTGCA）的寡核苷酸插入pgl3启动子载体的Bgl II和Mlu I位点。将构建的质粒载体转染到猪卵巢颗粒细胞中。颗粒细胞与猪卵母细胞一起在荧光素的存在下培养，荧光素酶的底物在FSH刺激下。卵母细胞的存在降低了FSH的作用，表明卵母细胞向颗粒细胞释放抑制物质。对成肌细胞向肌管分化的研究表明，Smad活性随着Smad响应元件的增加而增加。利用pGL3载体构建孕酮受体元件、雄激素受体元件和雌激素受体元件，并实时测定各自启动子活性。但是，该系统在荧光素酶半衰期短、质粒载体转染细胞效率高、抑制转染细胞凋亡等方面还有待进一步发展。

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.