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

本课题旨在制备α 2 A肾上腺素能受体（α 2 A AR）与绿色荧光蛋白（GFP）融合的转基因小鼠。通过修饰HA标记的载体（在α 2 A AR的起始密码子周围添加30 bp）制备靶向载体，所述载体由范德比尔特大学药理学系赠送。通过电穿孔将靶向载体转染到来源于小鼠品系129 SvEv的ES细胞中。经G418正负筛选后，PCR筛选出10个阳性菌落。Southern杂交显示3个阳性克隆。将两个克隆显微注射到C57 BL/6小鼠的胚细胞中。我们发现了3只雌性和2只雄性黑猩猩。F1杂合子小鼠交配产生41个杂合子型（20个雄性和21个雌性），没有纯合子。免疫组化结果显示，杂合子蓝斑中有GFP表达，但荧光强度不强。因此，我们正在尝试利用其他ES细胞系获得同型小鼠，虽然我们还没有发表这项研究，因为同型小鼠还没有得到，没有人能够产生这种类型的转基因小鼠。一旦获得同型小鼠，我们将立即进行生理分析，并考虑与其他研究所合作。此外，我们将应用多光子激发诱发的发色团辅助的激光失活来对抗右美托咪定（α 2激动剂）在该转基因小鼠中的麻醉。

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.