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

本研究的目的是产生用于人类精子功能诊断的透明带。我们试图制造表达猪ZPA蛋白的转基因小鼠，该蛋白与人类精子结合。我们之所以选择猪的ZPA而不是人的ZPA，是因为另一个研究小组已经开始使用人类ZPA基因进行类似的研究。利用小鼠ZPA启动子区（1.4kb）和猪ZPA全长DNA （6.8kb）和兔β-珠蛋白多聚腺苷化信号（0.5kb）构建了该转基因基因。小鼠ZPA启动子DNA片段由NIH inUA的j . dean博士提供。为了将转基因引入小鼠胚胎，在交配前用PMS/hCG处理BDF1雌性小鼠进行超排卵。将注射后的胚胎植入假孕小鼠输卵管内。用从尾部提取的DNA进行PCR分析，检测转基因的结合情况。在4只新生小鼠中检测到转基因。其中2只（_<2,bbb80）将转基因基因传给了后代。相应的转录本不仅在卵巢组织中检测到，而且在肝、心、肺等其他组织中也检测到。但蛋白仅在卵母细胞和透明带中表达。目前正在研究转基因小鼠透明带的异源精子结合能力。

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.