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

为了获得Na^+/I^—同调子的cDNA，我使用表达载体pCDM8和来自FRTL-5的分子量大于1kb的mRNA构建了cDNA文库。图书馆被分成120个池，每池有500到2000个大肠杆菌菌落。每个池纯化的质粒用葡聚糖法转染Cos7，测量^<125> i摄取。然而，在这个筛选系统中，我没有得到Na^+/I^- symporter的cdna。因此，我得出结论，Na^+/I^- symporter不会在Cos7中成功表达。下一步，我计划采用Vilijn的方法筛选Na^+/I^- symporter cDNA，即将体外每个质粒池转录的mrna注射到爪蟾卵母细胞中，测量其^<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并进行显微注射练习。因此，我没有Na^+/I^-同体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.