I. Genetic Operation Of Large Fragment In Chromosome II. Fast Selecting SiRNA Targeting Sites And ShRNA Transcript Controlling

The thesis is composed of six chapters. The first chapter is the in vivo excision and amplification of large human genomic segments using Cre-loxP and yeast artificial chromosome technology. The second chapter is the construction a library of human immunoglobin heavy chain gene using transformation-associated recombination (TAR) cloning method. The third chapter is the review of the latest literatures related with the gene targeting. The fourth chapter is a method of fast screening target sites for RNA interference using a cell-free system. The fifth chapter is a transactivated minimal E1b promoter driving the expression of short hairpin RNA. The sixth chapter is the review of the mechanism of RNA interference and appliance in gene therapy.Chapter I. In vivo excision and amplification of pre-determined large genomic segments, directly from the genome of a nature host, has been a powerful tool for obtaining genomic sequences of whole large gene. In this study, human L-02 cells were devised by combining the Cre/loxP system of yeast artificial chromosome. Two loxP sequences, each of which serves as recognition site for recombinase Cre, were integrated into Cα1 and V2-26 regions of the immunoglobulin heavy chain gene(hIgH) respectively by homologous recombination. Cell clones with circular YAC can be obtained after Cre recombinase action and puromycin selected. Analysized by Southern blot and FISH ,circular YACs containing hIgH gene cluster are maintained episomes under selective pressure . It is a very useful method for obtaining large genomic fragments directly from human cells without using foreign hosts. Therefore, our approach can be used effectively for gap sequencing of a genome, gene therapy, and functional analysis of unknown genes in human cells.