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.

记者基因测定是一个有用的系统，用于实时测量，用于在细胞到细胞，生殖细胞到单元细胞以及胚胎到胚细胞通信中起作用的旁分泌和自分泌因子的表达和动作时间。该系统由靶基因的调节域构建的荧光素酶基因载体（PGL3）组成，该靶基因被转染到卵巢细胞中，例如颗粒细胞，例如颗粒细胞，以及未分化的细胞，例如胚胎肌细胞。例如，构建了CRE报道矢量。简而言之，将包含3个共识CRE（TGACTGCA）串联的寡核苷酸插入了PGL3启动器载体的BGL II和MLU I位点。将Cre结构的质粒载体转染到从猪卵巢中分离的颗粒细胞中。在FSH刺激后，在荧光素A荧光素酶的底物存在下，将颗粒细胞用猪卵母细胞培养。卵母细胞的存在降低了FSH的作用，表明卵母细胞释放到颗粒细胞中。关于成肌细胞分化为肌管的研究表明，SMAD反应元素的SMAD活性增加。此外，孕激素受体元件，雄激素受体元件和雌激素受体元件类似地使用PGL3载体构建，并开发了各自启动子活性的实时测量。但是，应进一步开发该系统，用于荧光素酶的短期寿命，质粒载体转染效率到细胞的转染效率，并抑制转染细胞的凋亡。