| Now, DNA vector-based RNAi technology seemes to be the simple, economical, effective and safe way to introduce small interference RNA into mammalian cells. RNA polymeraseⅢ(PolⅢ) and RNA polymeraseⅡ(PolⅡ) promoters are used widely in vector. PolⅢpromoter can express the high levels of siRNA and mediate highly efficacious silencing. However, these promoters have no cell specificity and exceedingly high levels of siRNA expression increases the probability of off-target and elicit non specific effects such as interferon response and cellular toxicity. Comparing with PolⅢpromoters, PolⅡpromoters have cell or tissue specific and synthesizes some endogenous microRNA(miRNA). Therefore, using PolⅡto synthesize the siRNA of strategies mimic the natural miRNA synthesis and can be an efficient RNAi strategy in vivo.But, some studies show that using PolⅡto synthesize the siRNA for mimicking the natural miRNA synthesis have a lower silencing efficiency than the using PolⅢto synthesize the siRNA.Try to use PolⅡpromoter to express long double-stranded RNA(dsRNA ) is a very important area for researching the target tissue-specific gene silencing.Long dsRNA can be processed into 21-23 nucleotides(nt) small interfering RNA(siRNA) in the nucleus, which then moves to the cytosol to induce the siRNA effect. and it has the characteristics of multi-site interference effects. So that it has better the efficiency of RNA interference.some studies show that it will not produce off target effects. What is more, It is fit for recognizing and controlling by PolⅡpromoter. To achieve the purpose of improving the quality of animal products , transgenic technology is applied to produce specific gene silencing of organization and developmental stage. For example, cow milk has been used as infant formula for its universality of source, abundance of nutrition and the comparability between cow milk and human milk. However, There are still some important differences between them, which make the cow milk protein not be easily digested and absorbed, Infants will be caused allegic phenomenon by as1-casein andβ-lactoglobulin. If transgenic technology could produce to express special gene of long dsRNA in mammary epithelial cells, which effectively silence those gene.this will be effectively changed the composition of cow milk more suitable for infants drinking. Now, the gene knockout technology is still immature in livestock, so this idea has a higher possibility. But the nascent transcripts of hnRNA by PolⅡpromoters are transferred to the cytosol immediately after transcription, some studies show that dsRNA of >30bp will induce interferon response. Expression long dsRNA from the PolⅡpromoter to achieve the RNA interfence has a problem how blocks the long dsRNA in the nucleus before being processed into siRNA(less than 30bp), so preventing the export of long dsRNA to the cytosol for avoiding the interferon response.In this study, we also make some attempt.To explore the feasibility of applying RNA interference vector to express long dsRNA from the PolⅡpromoter for preventing the interferon response in mammalian cells. In the following aspects, we have carried out research: The eukaryotic expression cassette for RNA interference has been constructed using pcDNA3.1(+) as the vector. We design a self-cleaving ribozyme of HDV to cut off the 5'cap structure in transcription unit 5'end and a self-cleaving of hairpin ribozyme (tobacco ringspot virus) to remove the 3'polyA tail in the gene coding sequence 3'end. And BGHpA(Bovine Growth Hormone Polyadenylation Signal) is added to provide a polyadenylation signal for transcription termination in the downstream of hairpin ribozyme . In order to prevent the phenomenon of read-through ,we add a self-cleaving of hairpin ribozyme(tobacco ringspot virus) in the downstream of BGHpA. Eventually,we add a Humanβ-actin gene for delaying the RNA polymeraseⅡtranscription. To obtain a expression long dsRNA of cellline by the G418 screening , We have been transformed the entire structure to carrying the neomycin resistance gene of pBluescriptⅡSK (+) vector.To detect the founction primery of RNAi vector,We have added a reporter gene of enhanced green fluorescent protein (eGFP) in the vector and transfected PK15 cells to observe expression situation of the green fluorescent protein gene. The expression of green fluorescent protein is used to indicate the efficiency of the RNAi vector . and it easy to judge the preliminary function of the vector by green fluorescent. We set up a control group and experimental group,while transfect the PK15 cells. A larger than the RNAi vector has been constructed for eliminating the influence of vector size on the transfection efficiency. At the same time,expression of red fluorescent protein reporter gene vector is constructed for cotransfecting with these two groups. According to express of red fluorescent indicate the transfection efficiency of these two groups. The results show that the control group has been observed the green fluorescence and the experimental group has not been observed the green fluorescence in the same transfection efficiency. A preliminary study on the vector show that it can achieve the expectation. Subsequently, We apply semi-quantitative RT-PCR to detect the mRNA difference of GFP in each group.The result show that the experimental group is decrease by 46.1 percent than the control group, So the vector has played a certain effect on inhibition of mRNA export.In conclusion, we have already created a vector system for expressing mRNA to block in the nucleus. It has been applied to express long dsRNA from the PolⅡ promoter for avoiding long dsRNA export to induce the interferon responset and estimated its function at the cellular and RNA level. We have been obtained a vector system for reducing the mRNA export. This study laid the foundation for expressing long dsRNA from the PolⅡpromoter to prevent the interferon response in mammalian cells and studing of gene fuction and producing of transgenic animals.
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