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

通过生成催产素受体基因缺陷小鼠对催产素受体进行功能分析。

基本信息

批准号：
09660070
负责人：
NISHIMORI Katsuhiko
金额：
$ 2.43万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660070/
关键词：
Oxytocin Receptor G-protein linked knockout conditional tissues specific

项目摘要

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

在申请该基金后，Kimura博士（大坂大学）发表了小鼠OTR基因的序列。在此基础上，我们从1295 vEv小鼠脑组织cDNA中获得了OTR cDNA。利用该cDNA和pGKhprt/MCA 1 tk载体构建OTR靶向载体。就在这个载体完成后，我们从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，在一个实验室进行。在美国国立卫生研究院的研究中，他们得到了意想不到的结果：OTR缺陷纯合子小鼠在子宫内的早期胚胎死亡。在此基础上，我们将四种初始的“常规敲除OTR基因”策略改为“条件敲除”策略。条件敲除依赖于特异性元件loxP与来自P1噬菌体的细菌重组酶cre基因的结合。在建立ES细胞系后，其靶基因被“狐狸基因”同源取代，ES细胞用于产生嵌合体，并依次产生纯合突变小鼠。 ...更多信息将cre基因导入Tc-inducible启动子或组织特异性增强子/启动子调控下的转基因小鼠，与Tc-P小鼠杂交，可产生新的突变小鼠，其目的基因在特定组织或胚胎发育的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月开始开始这个合作项目。我们从KimuRa博士那里获得了携带小鼠OTR基因的λ噬菌体，并对OTR基因进行了详细的限制性酶切图谱分析，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和氨苄青霉素抗性基因的载体。该载体全长18.5kb。目前，我们正在将该载体导入E14 TG 2a ES细胞中，并筛选出OTR基因被正确靶向的细胞。在不久的将来，我们将使用新的诱导型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。该突变小鼠系统由Brown博士开发，C.in Baylor College of Medicine，Houston，Texas，USA。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cre-基因的突变小鼠。这种突变小鼠是由大坂大学的Miwa博士产生的。少