Construction Of Eukaryotic Vector Encoding Shrna Targeting GLT-1 Gene And Its Knocking-down Effects In Rat Glial Cells

Extracellular concentrations of the predominant excitatory neurotransmitter, glutamate, and related excitatory amino acids are maintained at relatively low level to ensure an appropriate signal-to-noise ratio and to prevent excessive activation of glutamate receptors that can result in cell death. Glutamate transporter is a kind of glycoprotein located in neurons and glial cell membrane, and it can quickly transport glutamate between synaptic cleft. It plays an important role in the signaling transduction between the normal synaptic and to avoid the toxic effects of Glu. Epileptic syndromes have diverse primary causes, including genetic background, develop-mental or acquired reasons. In rodent models, altering expression of glutamate receptor or glutamate transporter by knockout or knockdown procedures is culpable for enhancement or suppression of epileptic seizures. GLT-1 belongs to Glu Ts family, which contains five kinds of subtypes. GLT-1 expresses only in astrocytes and acts as the dominat transporter proteins in the brain. Recently, it was found that, the expression of GLT-1 was associated with the development and susceptibility of epileptic seizures.Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method inducing silence of gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi. Also, it is associated with regulatory processes, such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silence. The first step involves degradation of dsRNA into small interfering RNAs (siRNA), 21 to 23 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Some key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing noncoding RNAs, called microRNAs. The biogenesis and function of microRNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is tightly controlled. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.Objective:To construct a eukaryotic vector encoding shRNA targeting GLT-1,the coding information the plasmid was identified by DNA sequencing. Then, GLT-1 expression and cellular activities of glial cells transfected with thIS vector were determined. Thus, this work throws light on the mechanism of epilepsy, by producing novel methods and theory basis.Methods:Acorrding to the sequence of GLT-1, the hairpin siRNA templates were designed. The annealing product was then cloned into pSupressorNeo vector. The recombinant vector, pSuppressorNeo-GLT-1, was developed to express GLT-1 siRNA. RT-PCR, Western-blot, and immunofluorescence were performed to determing the expression of GLT-1 in rat glial cells transfected with pSuppressorNeo-GLT-1.Results:1. The recombinant plasmid pSuppressorNeo-GLT-1(Pa,Pb,Pc)were successfully constructed, identified by restricted enzyme digestion and DNA sequencing.2. The three siRNA expression plasmids pSuppressorNeo-GLT-1(Pa,Pb,Pc)were transfected into rat glial cells by lipofectamine, respectively. They significantly decreased the expression levels of GLT-1 in transient transfected rat glial cells detected by RT-PCR , Western-blot, and immunofluorescence. Intriguingly, the effects of pSuppressorNeo-GLT-1 Pa and Pb were the more remarkable than that SuppressorNeo-GLT-1 Pc.Conclusion : Using RNA interference techniques, we successfully constructed the recombinant plasmid pSuppressorNeo-GLT-1. The expression level GLT-1 was significantly decreased in rat glial cells transfected with the recombinant plasmid pSuppressorNeo-GLT-1. Our work underscores the importance of research on the relationship between GLT-1 protein and epilepsy. Importantly, we found that effective siRNA of GLT-1 will give clues to a perspective therapeutic strategy against epilepsy.