Novel strategies for studying gene and protein function: Random shRNA library synthesis and Transposon Directed Base-Exchange Mutagenesis

With the completion of the human genome sequence, about 30,000 genes have been identified. However, the function of many genes remains unknown. Therefore, much effort has focused on identifying the biological function of given genes. Recently RNA interference (RNAi) has emerged as an important research tool to assay gene function within the mammalian cell. In order to identify genes responsible for a particular phenotype using RNAi, many different types of libraries have been tested and successfully used. However, there has been no report of a completely random RNAi library. In order to identify novel gene functions, we have now developed a random 18 base RNAi library.;Any newly identified protein must be further studied in order to understand how its novel functions and properties derive from its amino acid sequence. This goal can be achieved through mutagenesis by observing the altered phenotype in a library of mutants. Many mutagenesis technologies have been developed in last three decades to determine the relationship between sequence, structure and function. Mutagenesis technologies also have been successfully used to alter the original function of proteins. However, all of these mutagenesis methods have specific advantages and disadvantages. In order to develop a more efficient mutagenesis method, we developed a novel mutagenesis method, Transposon Directed Base-Exchange Mutagenesis (TDEM) that uses a transposon to create a controllable mutant library.;For efficient TDEM, we needed improved transposition activity so that all possible mutations could be obtained. Therefore, we developed a new transposition system using N-terminally deleted MuA, which shows more robust transposase activity. This system was further optimized by testing different buffer compositions.;The above-mentioned random RNAi library and TDEM require many tedious plasmid purification steps. Therefore, in order to achieve more rapid and economic plasmid purification, we developed an efficient plasmid purification method using glass syringe filters.