Plasmid-based SiRNA Silencing Of GATA-1 Gene Expression

The process of Hematopoiesis is tightly controlled by various oineage-specific transcription factors.Among them,GATA transcription factors are one of the most well-studied transcription factors.The GATA-1 has been detected in erythroid,megakartocytic,eosinophilic,and mast cell lines within the hematopoietic system.,particularly,the requirement for GATA-1 was different between the proliferation and differentiation of primitive erythropoiesis that only a short duration of GATA-1 expression was indispensable for the full maturation of erythroid production,generates nucleated,primitive erythrocytes(EryPs).The studies on various transcription factors involved in hematopoietic development and differentiation has been extensively investigated,manly using gene-manipulated murine embryos or embryonic stem(ES) cells.Regarding to primates(monkey and human),however,regulative mechanisms remains to be elucidated due to ethical limitation of using their embryos or fetus.Although primate ES cells will be expected due to serve as useful tool for such studies,it is difficult to conduct efficient gene-manipulation by using the same method as murine ES cells.RNA interference(RNAi) is a mechanism,whereby double-stranded RNA (dsRNA) directs sequence-specific gene silencing via degradation of homologous RNAs,translational inhibition or chromatin modification.Using short(<30bp)small intering dsRNAs(siRNA) circumvents non-sequence-specific responses to longer dsRNAs,and allows sequence-specific mRNA targeting in mammalian cells.Recently we have established a differentiation induction system in which erythrocytes,megakaryocytes ,and neutriphils are generated from cynomolgus monkey ES(CES) cells by co-culture with OP9 stromal cells.This siRNA system may become a good tool to evaluate the contribution of GATA-1 gene during CES cell differentiation.The small interference RNA( siRNA) has already widely used for silence of gene expression in various organisms. Here,we have investigated that siRNA suppress the target gene(GATA-1)'s expression in K562 cells using pBA-hU6-Neo vector. Then confirmating the repression of GATA-1 in modified K562 cells.We examine the silence of GATA-1 gene in K562 cells. This siRNA system may become a good tool to study the mechanism of the effection of GATA-1 gene silence on hematopoietic function.This research uses special siRNA,which repress the expression of GATA-1 in mRNA level .It makes a beneficial tool for the further studying of GATA-1 gene's function and possible molecular mechanism in building blood. The research includes the contents as follows:We examine GATA-1 gene by PT-PCR,test the level of gene expression with GAPDH mRNA as a control,observe the expression of the GATA-1 gene.The siRNA's special repression to RNA is a gene silent phenomenon induced by double-stranded RNA. It can decline the particular mRNA,which makes it can't be translated into protein, thus repressing the expression of the particular gene.This research produce siRNA through siRNA expression vector and the expression frame of siRNA produced by PCR,and then transfect K562 cells that express GATA-1 gene by electroporation ,and then culture the K562 cells.After that we examine the transfection efficiency and the repression of siRNA to GATA-1 by RT-PCR.It tests the sepicality of RNAi using siRNA which has three mutable sites as a control.There is no similar research reported at home and abroad. The result shows that siRNA can be transfected into K562 cells efficiencially by electroporation, the efficiency is above 80%.The siRNA GATA-1 can repress the expression of the GATA-1gene in K562 cells.The metered RT- PCR examination result shows that the siRNA can reduce the level of mRNA GATA-1 by about 80%( 24 hs after transfection) and 90%( 10 ds after transfection).The experiment proved that siRNA can repress the expression of the gene GATA-1 efficiently and specially, which supplys a valuable method for studying GATA-1 gene function and its downstream signals.