The Establishment Of Stable Integration Of Cellular And Animal Level Of Rna Interference Technology And Mouse Globin Expression In The Regulation Of The Use

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). This phenomenon has been found in plants, trypanosome, planaria, hydra, drosophila, fungi, caneorhabditis elegans, zebrafish, mouse embryo. Short interfering RNA (siRNA) is the heart of RNAi. Long dsRNA can be processed to siRNA of 21-23 nucleotides (nt) by Dicer, a RNA III -type endonuclease. Upon binding to RNA-induced silencing complex (RISC), double-stranded siRNA is unwound and targeted to homologous mRNAs, resulting in sequence-specific cleavage and degration of mRNA. In mammalian cells, dsRNA longer than 30 nt can trigger interferon response via activation of PKR, leading to a global inhibition of protein synthesis. This view changed in 2001, with the crucial insight of Tuschl and colleagues, they showed that chemically synthesized siRNA molecules of 21-22 nt could be used to target mammalian genes by RNAi while evading the interferon response, which provided a new way for RNAi used in mammalian system.More and more researchers are using siRNAs to reduce the expression of specific mammalian genes. Currently, there are five methods for generating siRNAs for gene silencing studies: 1. Chemical synthesis; 2. In vitro transcription; 3. Digestion of long dsRNA by an RNase III family enzyme; 4. Expression in cells from an siRNA expression plasmid; 5. Expression in cells from a PCR-derived siRNA expression cassette. However, these systems rely on transfection for delivery and cannot generate long-term gene silencing in vivo. This obstacle may be circumvented by recently developed retrovirus- and lentivirus-delivered RNAi.The retrovirus is the most commonly used because of its high transduction efficiency, stable insertion of a gene into host genome, and lack of adverse host immune response to retroviral vectors. In this system, GP-293 cell line which can stably express the retrovirus proteins, gag and pol, is used as a panphotropic packaging cell line.The murine α- and β-globin loci are multigene clusters located on chromosomes 11 and 7 respectively. The murine a locus consists of three genes (ζ , α1 and α2). The murine β locus consists...