Functional analysis of oxytocin receptor by generation of oxytocin receptor gene deficient mice.

在申请这项资助后，木村博士（oo坂大学）发表了小鼠OTR基因的序列。基于该序列，我们从1295只vEv小鼠脑源性cDNA中获得了OTR cDNA。利用该cDNA和pGKhprt/MCA1tk载体构建otr靶向载体。就在这个载体完成后，我们从我在美国的朋友那里得到了一个重要的信息，说一个实验室正在进行OTR基因的敲除项目。在美国国立卫生研究院，给了他们意想不到的结果：OTR缺陷纯合子小鼠在子宫内的早期胚胎死亡。根据这些信息，我们将四种最初的“常规敲除OTR基因”策略改为“条件敲除”策略。条件敲除依赖于特定元件loxP和基因编码细菌重组酶cre的结合，该基因编码来自于P1噬菌体。建立目的基因被“狐狸基因”同源替代的胚胎干细胞系后，利用胚胎干细胞培育嵌合、纯合突变小鼠。在tc诱导启动子或组织特异性增强子/启动子的控制下，将携带外源cre基因的floxP小鼠与其他转基因小鼠杂交，可以产生新的突变小鼠，其靶基因在胚胎发生的特定组织或特定阶段丢失或失活。由于OTR基因敲除实验情况的改变，我和木村博士决定从1998年4月开始合作开展这个项目。我们从KimuRa博士那里获得了携带小鼠OTR基因的lambda噬菌体，并绘制了详细的OTR基因限制性内切图谱，生成了新的OTR敲除载体。该载体包括新霉素耐药基因、3个loxP元件、OTR基因5′臂和3′臂、编码外显子2和3的片段、HSV-tk基因，以及编码ori和氨苄西林耐药基因的载体。这个新向量的总长度为18.5 kb。现在我们将该载体导入E14TG2a ES细胞中进行筛选，其OTR基因被正确靶向。在不久的将来，我们将使用新的诱导cre系统，在该系统中，突变的类固醇受体-cre融合蛋白将被类固醇衍生物激活。这种突变小鼠系统是由美国德克萨斯州休斯顿贝勒医学院的布朗博士开发的。对于子宫特异性OTR基因敲除，我们将获得在平滑肌特异性肌动蛋白启动子控制下携带cree基因的突变小鼠。这只突变小鼠是由大阪大学的Miwa博士培育的。少

After the application of this grant, Dr. Kimura (Oosaka-Univ) published the sequence of mouse OTR gene. Based on that sequence, we got OTR cDNA from brain derived cDNA of 1295 vEv mouse. With this cDNA and pGKhprt/MCA1tk vector, we generated OTR-targeting vector. Just after the completion of this vector, we got an important information from my friend in USA, saying that knock-out project for OTR gene, carried out at a Lab. in NIH, USA, gave them unexpected result : the early embryonic death of OTR deficient homozygous mice in utero. With this information, we changed four initial strategy of "conventional knockout of OTR gene" to "conditionalknockout". The conditional knockout are dependent of combination of specificelement loxP and the gene coding bacterial recombinase cre, derived from P1 phage. After establishing of ES cell line, whose targeted gene is homologousely substituted by "fox gene", the ES cells are used to generate the chimeric, and successively the homozygous mutant mice. … More Crossing of floxP mice with the other trasgenic mice, which is harboring exogenous cre gene under control of Tc-inducible promoter, or tissue specific enhancer/promoter, can generate new mutant mice, whose targeted gene is lost and inactivated in specific tissue, or specific stage in embryogenesis. Because of the alteration of experimental situation about OTR gene knockout, I and Dr. Kimura decided to begin this project as a collaboration from April, 1998. We gotlambda phage harboring mouse OTR gene from Dr. KimuRa, and made detailed restriction maps of OTR gene, and generated new OTR knockout vector. This vector comprises neomycin resistant gene, 3 loxP elements, 5'arm and 3'arm of OTR gene, the fragment coding for exon 2 and 3, HSV-tk gene, and vector encoding ori andampicillin resistant gene. The total length of this new vector is 18.5 kb. Now we are introducing this vector to E14TG2a ES cell and screening the cell, whose OTR gene was correctly targeted. In near future, we will use new inducible cre system, in which mutated steroid receptor-cre fusion protein will be activated by the administration of steroid derivative. This mutant mouse system is being developed by Dr. Brown, C.in Baylor College of Medicine, Houston, Texas, USA.For uterus specific OTR geneknock out, we will obtain a mutant mouse which is harboring cre-gene under control of smooth muscle specific actin promoter. This mutant mouse was generated by Dr. Miwa, Osaka university. Less