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

荧光素酶报告基因卵巢生物调节剂实时检测系统的研制及其应用

基本信息

批准号：
16380200
负责人：
HATTORI Masa-aki
金额：
$ 9.73万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

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.

报告基因测定是一种有用的系统，可实时测量在细胞间、生殖细胞与体细胞以及胚胎与体细胞通讯中起作用的旁分泌和自分泌因子的表达和作用时间。该系统由构建有目标基因调控域的荧光素酶基因载体（pGL3）组成，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，构建了CRE报告载体；简而言之，将含有 3 个共有 CRE (TGACTGCA) 串联的寡核苷酸插入 pGL3 启动子载体的 Bgl II 和 Mlu I 位点。将CRE构建的质粒载体转染至从猪卵巢分离的颗粒细胞中。在 FSH 刺激后，在荧光素（一种荧光素酶的底物）存在下，将颗粒细胞与猪卵母细胞一起培养。卵母细胞的存在降低了 FSH 的作用，表明卵母细胞将抑制物质释放到颗粒细胞中。对成肌细胞分化为肌管的研究表明，Smad 反应元件的 Smad 活性增加。此外，用pGL3载体类似地构建孕激素受体元件、雄激素受体元件和雌激素受体元件，并开发了各自启动子活性的实时测量。但荧光素酶的半衰期短、质粒载体转染细胞的效率以及转染细胞凋亡的抑制作用还需要进一步开发。