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

在这项资助申请后，木村博士(大阪大学)发表了小鼠OTR基因的序列。根据该序列，我们从1295只VEV小鼠的脑组织中获得了OTRcDNA。将该基因与pGKhprt/MCA1tk载体构建成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OTR基因的敲除项目是在实验室进行的。在美国国立卫生研究院，他们给了他们意想不到的结果：OTR缺陷纯合子小鼠在子宫中的早期胚胎死亡。有了这些信息，我们将四种最初的“常规基因敲除”策略改为“条件基因敲除策略”。条件性基因敲除依赖于特异性元件loxP和编码细菌重组酶cre的基因的组合，该基因来源于P1噬菌体。目的基因被狐狸基因同源替换的ES细胞系建立后，用ES细胞产生嵌合体，继而产生纯合子突变小鼠。…在组织特异性启动子或组织特异性增强子/启动子控制下携带外源cre基因的转基因小鼠与FloxP小鼠杂交更多，可以产生新的突变小鼠，其靶基因在特定组织或胚胎发育的特定阶段丢失和失活。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月开始合作开展这一项目。我们从木村博士那里获得了携带小鼠OTR基因的噬菌体，并绘制了详细的限制性内切酶图谱，构建了新的OTR基因敲除载体。该载体包括新霉素抗性基因、3个loxP元件、OTR基因的5‘臂和3’臂、编码外显子2和3的片段、HSV-tk基因和编码ori和氨苄西林抗性基因的载体。该新载体全长18.5kb。现在我们正在将该载体导入E14TG2a ES细胞，并筛选出其OTR基因被正确靶向的细胞。在不久的将来，我们将使用新的可诱导的cre系统，其中突变的类固醇受体-cre融合蛋白将通过给予类固醇衍生物来激活。这个突变小鼠系统是由美国德克萨斯州休斯敦贝勒医学院的Brown，C.博士开发的。对于子宫特异性OTR基因敲除，我们将获得一个在平滑肌特异性肌动蛋白启动子控制下携带cre基因的突变小鼠。这只突变的小鼠是由大阪大学的Miwa博士培育的。较少

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