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

人ZPA转基因小鼠的制备及其在精子功能诊断中的应用

基本信息

批准号：
10470350
负责人：
KOYAMA Koji
金额：
$ 7.81万
依托单位：
Hyogo college of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10470350/
关键词：
Zona pellucida Pig ZPA Transgenic mice Male infertility Sperm function diagnosis In vitro fertilization ZPA 受精受精能獲得精子レセプター先体反応不妊症

项目摘要

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.

本研究的目的是制备用于人类精子功能诊断的透明精子。我们试图制造转基因小鼠，表达猪ZPA蛋白，人类精子结合。我们选择猪ZPA而不是人类的原因是另一个小组已经开始了使用人类ZPA基因的类似研究。以小鼠ZPA基因启动子区（1.4kb）和猪ZPA基因全长（6.8kb）及兔β-珠蛋白多聚腺苷酸化信号（0.5kb）为微基因，构建了转基因小鼠ZPA。小鼠ZPA基因启动子DNA片段由美国国立卫生研究院（NIH）的Dr.J.Dean在UA提供，为了将转基因导入小鼠胚胎，在交配前用PMS/hCG处理BDF 1雌性小鼠进行超数排卵。将注射的胚胎植入假孕小鼠的输卵管中。使用从尾部提取的DNA通过PCR分析检查转基因的掺入。在四只新生小鼠中检测到转基因。其中2株（<2，>8）将转基因转入后代。相应的转录本不仅在卵巢组织中检测到，而且在其他组织如肝、心、肺中也检测到。而蛋白质仅在卵母细胞和透明质细胞中表达。转基因小鼠卵透明带的异源精子结合能力目前正在研究中。