Production of transgenic mice expressing human ZPA and its application to sperm function diagnosis

本研究的目的是制备用于人类精子功能诊断的透明带。我们试图制造表达人精子结合的猪ZPA蛋白的转基因小鼠。我们之所以选择猪ZPA而不是人，是因为另一个小组已经开始了使用人ZPA基因的类似研究。构建了含小鼠ZPA启动子区域(1.4kb)、猪ZPA全长基因(6.8kb)和兔β-珠蛋白多聚腺苷化信号(0.5kb)的转基因载体。小鼠ZPA启动子DNA片段由美国国立卫生研究院院长J博士亲切提供。为了将转基因导入BDF1雌性小鼠胚胎，在交配前用PMS/hCG进行超数排卵。将注射的胚胎植入假孕小鼠的输卵管内。用从尾巴中提取的DNA进行PCR分析，检测转基因的掺入情况。在四只新生的小鼠身上检测到了转基因。其中两个(_&lt；2，&gt；8)将转基因基因转移到了它们的后代。不仅在卵巢组织中检测到相应的转录本，在肝、心、肺等其他组织中也检测到相应的转录本。但仅在卵母细胞和透明带中观察到蛋白表达。转基因小鼠透明带的异源精子结合能力目前正在研究中。

The objective of this study is to produce zona pellucida for use in human sperm function diagnosis. We attempted to make transgenic mice expressing pig ZPA protein to which human sperm binds. The reason why we selected pig ZPA instead of human is that another group had already started a similar study using the human ZPA gene. The transgene was constructed with the promoter region of mouse ZPA (1.4kb) and the minigene including pig ZPA full-length DNA (6.8kb) and polyadenylation signal of rabbit β-globin (0.5kb). The promoter DNA fragment of mouse ZPA was kindly provided by Dr.J.Dean of NIH inUA.In order to introduce the transgene into mouse embryos BDF1 female mice were treated with PMS/hCG for superovulation before mating. The injected embryos was implanted in the oviducts of pseudo-pregnant mice. The incorporation of the transgene were examined by PCR analysis using DNA extracted from the tails. The transgene was detected in four newly born mice. Two (_<2, >8) of them transferred the transgene genes to their progenies. The corresponding transcripts were detected not only in ovarian tissue but also in other tissues including liver, heart, lung. But protein expression was only observed in oocytes and zona pellucida. The heterologous sperm binding capacity of the zona pellucida from the transgenic mice is currently being investigated.