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Making a transgenic mouse with α 2A adrenoceptor fused with GFP

制作α2A肾上腺素受体与GFP融合的转基因小鼠

基本信息

批准号：
15390478
负责人：
MIZOBE Toshiki
金额：
$ 9.28万
依托单位：
Kyoto Prefectural University 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：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

项目摘要

This project is to make transgenic mouse with alpha2A adrenergic receptor (alpha2A AR) fused with green fluorescent protein (GFP). The targeting vector was made by modifying the HA tagged vector (30 bp was added around the initiation codon of the alpha2A AR) which had been gifted from the Department of Pharmacology, the Vanderbilt University. The targeting vector was transfected by electropolation into the ES cell derived from mouse strain 129 SvEv. After positive-negative selection by G418, PCR screening showed 10 positive colonies. The Southern blotting revealed 3 positive clones. Two clones was micro-injected into blastcyte of C57BL/6 mouse. We got 3 female and 2 male chimerae. Mating of Fl hetero-mouses resulted in 41 hetero type (20 males and 21 females) with no homozygotes. Immunohistochemistry revealed that GFP was expressed in locus coeruleus in the heterozygotes, though the fluorescent intensity was not so strong. Therefore, we are trying to get homo-type mouse by using the other ES cell lines.Although we have not yet published about this study because homo-type mice have not yet got, no one has ever been able to produce this type of transgenic mouse. We are going to perform the physiological analysis as soon as getting homo-type mouse, and to consider the collaboration with other institutes. Also, we are going to apply the multiphoton excitation-evoked chromophore-assisted laser inactivation to antagonize the anesthesia by dexmedetomidine (alpha2 agonist) in this transgenic mouse.

本项目是将alpha2A肾上腺素能受体（alpha2A AR）与绿色荧光蛋白（GFP）融合的转基因小鼠。靶向载体由美国范德比尔特大学药学系提供的HA标记载体（在alpha2A AR起始密码子周围增加30 bp）进行修饰而成。将目标载体电转染至小鼠株129 SvEv胚胎干细胞中。经G418阳性阴性筛选，PCR筛选出10个阳性菌落。Southern印迹显示3个阳性克隆。将两个克隆微注射到C57BL/6小鼠胚细胞中。我们有3只雌性和2只雄性嵌合体。杂种小鼠共获得41个异型，其中雄性20个，雌性21个，无纯合子。免疫组化结果显示，GFP在杂合子蓝斑表达，但荧光强度不强。因此，我们正试图利用其他胚胎干细胞系获得同型小鼠。虽然我们还没有发表这项研究，因为同源型的老鼠还没有得到，但从来没有人能够生产出这种类型的转基因老鼠。我们将在获得同型小鼠后立即进行生理分析，并考虑与其他研究所的合作。”此外，我们将应用多光子激发诱发的发色团辅助激光失活来对抗右美托咪定（alpha2激动剂）对转基因小鼠的麻醉。