Foundation Of RNA Interference Expression System And Research Of CR-1 Gene Function

Since the discovery that synthetic double-stranded 21-nt small interfering RNA is enough to trigger gene-specific silencing, double-strand RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants, Caenorhabditis elegans, Drosophila and mammalian cells. However, we expect that a approach should be adaptable to situations for which delivery of in v/fro-synthesized siRNAs may not be practical, such as primary cell cultures, studies in intact animals, and gene therapy. In addition, to date, the most powerful applications of genetic manipulation are realized only with the creation of stable mutants.Here, a vector-based siRNA expression system is reported that can induce robust inhibition of endogenous or exterior gene stably. Using this system, various types of siRNA synthesized from DNA Templates in Vivo against GFP or CR-1 gene were tested on their induction of the sequence-specific RNA interference activity in human cells. Results indicate that the RNA secondary structure is associated with the RNAi activity. These findings highlight the general utility of this DNA vector-based RNAi technology in suppression gene suppression gene expression in mammalian cells and the applications in gene therapy.Cell regulator-1(CR-1) is a noval gene identified by Dr.Yiguang Wang recently and has been submitted to Genebank (Accession no.GI-21703347). With the result of yeast two-hybrid and the discovery that expression of CR-1 is rich in many tumor cell lines rather than normal cell lines, it is supposed to be involved in the initiation of tumor.Using this vector-based siRNA expression system, down-regulation of the CR-1 expression on 7721 cells by RNAi reversed the malignant morphometricsand exhibited a relatively slow growth rate compared with wild-type 7721 cells. Then the cell-cycle distribution is analyzed by flow cytometry, reducing CR-1 gene expression induced 7-fold increase of cells with sub-G1 content DNA compared with the control. Our study suggests that CR-1 gene is involved in the control of cellular proliferation and differentiation in the regulatory web in the cell cycle through the interaction with p21.