DEVELOPMENT OF EXPRESSION CLONING SYSTEM FOR ONCOGENE CDNAS

我们正在开发用于分离的cdna表达克隆系统。 显性癌基因和隐性癌基因。在这个系统里。Cdna 文库在真核表达载体中使用 Poly(A)-从转化子或肿瘤中选择的RNA。图书馆 然后，DNA可以用来转染NIH/3T3细胞。CDNA克隆将 从产生的病灶中恢复，并分析它们的结构。 为了使这一目的的表达克隆可行， 包含完整编码序列的文库将是必要的。 在这一点上，我们开发了一种高效的cDNA克隆 在真核生物中可以指导插入片段定位的系统 具有较大克隆能力的表达载体。我们的 含有逆转录病毒长末端的Lambda-质粒复合载体 重复(LTR)启动子在猴病毒40(SV40)中表达 早期启动子驱动的neo基因作为真核细胞的选择 记号笔。从含有该基因的接头-引物中合成了cdna。 Sfii的站点，这是一种罕见的DNA切割工具。连接了一个适配器 在双链cdna分子的两端，被 SfiI，并与切割制备的Lambda载体臂进行结扎 在两个不同的Sfii站点。由于定向克隆 策略与两端粘性的非对称结构 载体和插入DNA，效率为1000万到1亿 噬菌体的空斑形成单位从一微克的 Poly(A)-选择的RNA。此外，我们已经表明，我们的 文库包含多种生长因子和受体的cDNA 这些分子几乎是全长的2.5到6.5kb的分子 频率。

We are developing a cDNA expression cloning system for isolation of dominant as well as recessive oncogenes. ln this system. cDNA libraries are constructed in eukaryotic expression vectors using poly(A)-selected RNAs from transformants or tumors. The library DNA can then be used to transfect NIH/3T3 cells. cDNA clones will be recovered from resulting foci, and their structures analyzed. In order to make expression cloning feasible for this purpose, cDNA libraries containing complete coding sequences will be necessary. At this point, we have developed a high efficiency cDNA cloning system which can direct the orientation of inserts in eukaryotic expression vectors which possess large cloning capacities. Our lambda-plasmid composite vectors contain a retroviral long terminal repeat (LTR) promoter to express cDNA and a simian virus 40 (SV40) early promoter-driven neo gene as a eukaryotic cell selection marker. cDNA was synthesized from a linker-primer containing the site for SfiI, an infrequent cutter of DNA. An adaptor was ligated at both ends of the double-stranded cDNA molecules, cleaved by SfiI, and ligated with the lambda vector arms prepared by cutting at two different SfiI sites. Due to the directional cloning strategies and non-symmetrical structure of sticky ends of both the vector and insert DNAs, the efficiencies of 10 to 100 million plaque-forming units of phages were obtained from one microgram of poly(A)-selected RNA. Furthermore, we have shown that our libraries contain cDNAs for several growth factors and receptors that are nearly full length molecules of 2.5 to 6.5 kb, at high frequencies.