Manipulating The Gene Expression Through Competitive Inference Of Transcription Factor Binding Sites

Huge amount sequence information provided to researchers by the human genome project has substantially changed our knowledge base related to the development of diseases and more and more attention are being paid to the roles of genes in the disease processes.Various new technologies for the study of gene function are emerging,such as RNA interference technology,antisense technology,and genome editing technology.They are all in the level of gene regulation with various deficiencies.The gene products are usually proteins,while some RNAs,also gene products,have important function in the regulation of protein coding genes.The regulation of gene expression is an essential process to all known organisms.The steps needed to regulate gene expression including transcription,RNA splicing,translation and post-translational modification of proteins,among of which the basis of gene expression is transcription.This thesis work attempted to develop a novel method to manipulate gene express based on interference of transcription factor binding sites.As a test bed,we first confirmed the expression information of disease related genes in existing lab cell lines by real time PCR with designed primers targeting to their mRNA sequence.PCR and gel electrophoresis were used to verify the expression of the gene.We designed competing oligo probes with identical sequence of transcription factor binding sites of the target genes.The competing oligo probes were s used to interfere the expression of the target gene.Two groups of probes were designed as control,one with mutant sites and the other containing unrelated sequence that does not exist in the human genome.Probes were labeled with Cy3 fluorescent dye and then were transfected into cells.After the probe entered the cells,total RNA was extracted and the first strand of cDNA was synthesized as the template for qPCR amplification to verify the difference in gene expression.Our study demonstrated that,compared with the control groups,the expression of target gene in test group was evidently decreased by the detection of Real-time PCR.Our results,although preliminary,clearly indicated the feasibility of manipulating the gene expression level through competitive inference of transcription factor binding sites.Further development of method could offer a powerful tool with broad prospects for research and clinical application.