pIRES2-EGFP-DAZ3质粒构建及金黄地鼠2-细胞胚中人DAZ基因的复制与表达

背景与目的：人Yq11.2上AZF区 (内含DAZ基因家族) 的缺失是多数男性不育的病因。本研究旨在探讨DAZ3基因导入精子的可能性以及导入基因在2-细胞胚中的功能。材料与方法：构建pIRES2-EGFP-DAZ3质粒，经鉴定无误后将其导入金黄地鼠精子。将携带DAZ3基因的精子与金黄地鼠卵母细胞进行体外受精。用FISH、PCR、RT-PCR技术分别检测DAZ3基因在精子头部、雄原核上的整合以及在2-细胞胚中的复制与表达。结果：构建的pIRES2-EGFP-DAZ3质粒经酶切、PCR和绿色荧光蛋白表达鉴定构建成功。采用FISH在精子头部、单细胞胚雄原核和2-细胞胚两个间期核上观察到阳性杂交信号。采用RT-PCR在2-细胞胚样本中观察到DAZ3 cDNA的特异条带。结论：外源性DAZ3基因能被导入精子基因组。携带DAZ3基因的精子可与卵母细胞完成正常的受精过程。导入的DAZ3基因在早期胚胎细胞中能够随细胞分裂自我复制并表达其功能。本研究结果为DAZ基因缺失所致男性不育的治疗提供了新的线索。

Construction of the plRES2-EGFP-DAZ3 Plasmid and Replication and Expression of Human DAZ gene in Two- Cell Embryo of Golden Hamster

BACKGROUND & AIM: Deletions of the AZFc region in Yq11.2, including the DAZ gene family, are responsible for most cases of male infertility. The purpose of this study was to explore the feasibility of introducing DAZ3 gene into spermatozoa and the function of introduced gene in two-cell embryo. MATERIAL AND METHODS: pIRES2-EGFP-DAZ3 plasmids were constructed, identified and then introduced into spermatozoa of golden hamster. In vitro fertilization between the hamster oocytes and spermatozoa carrying DAZ3 gene were performed. Fluorescent in situ hybridization (FISH), PCR and RT-PCR were done to detect the integration of DAZ3 gene in sperm head, male pronuclei and to ascertain the replication and expression of DAZ3 gene in two-cell embryos. RESULTS: The successful construction of pIRES2-EGFP-DAZ3 plasmids were confirmed by the identification of endonuclease cutting, PCR and expression of green fluorescent protein. FISH showed the positive signals in the male pronuclei of one-cell embryos and each nucleus of two-cell embryos as well as in sperm heads. RT-PCR demonstrated the specific bands of DAZ3 cDNA in two-cell embryos. CONCLUSION: The exogenous DAZ3 gene could be introduced into sperm genome. The sperm carrying DAZ3 gene could complete the fertilization with oocyte normally. The introduced DAZ3 gene was able to replicate itself and express its function in early embryonic cells. Our results provided a new clue for the therapy of male infertility involving DAZ gene deletion.