Cloning and expression of Na^+/I^--symporter

为了获得Na^+/I^-同向转运体的cDNA，我使用表达载体pCDM 8和来自FRTL-5的分子大小大于1 kb的mRNA构建了cDNA文库。将文库分成120个池，由500至2，000个大肠杆菌菌落组成。通过葡聚糖方法将从每个池中纯化的质粒转染Cos 7以测量3 <125>I-摄取。但在此筛选系统中未能得到Na^+/I^-同向转运体的cDNA，因此，我认为Na^+/I^-同向转运体在Cos 7中不能成功表达。作为下一步的尝试，我计划用Vilijn的方法筛选Na^+/I^-同向转运体的cDNA，即从体外转录的每个质粒库中提取mRNA，注射到爪蟾卵母细胞中，以测定它们对^<125>I的摄取。因为我没有显微注射的工具，也没有显微注射的经验。我一直在购买显微注射工具和实践显微注射。因此，我没有关于Na^+/I^-同向转运体cDNA的数据。

In order to get a cDNA for Na^+/I^--symporter, I constructed a cDNA library using a expression vector, pCDM8, and mRNA with more than 1 kb molecular size from FRTL-5. The library was divided into 120 pools consisting of 500 to 2,000 E.coli colonies. The plasmid purified from each pool was transfected Cos7 by the dextran method to measure ^<125>I-uptake. However, I could not get any cDNAs for Na^+/I^--symporter in this screening system.Cosequently, I concluded that Na^+/I^--symporter would not express successfully in Cos7.As a next attempt, I have made a plan that cDNA for Na^+/I^--symporter should be screened by the procedure of Vilijn, i.e.mRNA transcribed from each plasmid pool in vitro should be injected into Xenopus oocytes to measure their ^<125>I uptake. Since I have no tools for microinjection and have had no experience in microinjection. I have been purchasing microinjection tools and practicing microinjection. Therefore, I have no data about Na^+/I^--symporter cDNA.