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

报告基因分析是实时测量细胞间、生殖细胞与体细胞、胚胎与体细胞通讯中旁分泌和自分泌因子的表达和作用时间的有用系统。该系统由带有靶基因调控结构域的荧光素酶基因载体(PGL3)组成，并将其导入卵巢细胞(如颗粒细胞)和未分化细胞(如胚胎成肌细胞)。例如，构建了Cre报告载体；简而言之，将含有3个共有Cre片段的寡核苷酸(TGACTGCA)插入到pGL3-启动子载体的BglⅡ和MLU I位点。将构建的Cre载体导入分离的猪卵巢颗粒细胞。颗粒细胞与猪卵母细胞在荧光素酶底物荧光素的存在下培养，以刺激FSH。卵母细胞的存在降低了卵泡刺激素的作用，提示卵母细胞向颗粒细胞释放抑制物质S。成肌细胞分化为肌管的研究表明，随着Smad反应元件的增加，Smad活性增加。此外，用pGL3载体构建了孕激素受体元件、雄激素受体元件和雌激素受体元件，并建立了相应启动子活性的实时检测方法。但由于荧光素酶的半衰期较短，质粒载体对细胞的转染率较高，抑制了细胞的凋亡，该系统还有待于进一步开发。

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.