Two-color Flurescence Technique Platform Of RNAi And Its Application Against SARS-CoV

RNA interference (RNAi) is short RNA-targeted degradation ofhomologous RNA. It can effectively and specifically knockdown bothendogenous and exogenous gene expression. Great efforts had beenmade in the application of RNAi technology to treat virus infectiousdiseases, especially AIDS, type B and C hepatitis, resulting in inspiringprogress.In the late of 2002, the severe acute respiratory syndrome (SARS),caused by the SARS-CoV, mysteriously developed in mainland ofChina and was spread to other areas worldwide. To response, manygroups devoted themselves to the research of anti SARS-CoV agent,including antibody, DNA vaccine, and so on. Two groups hadsuccessfully used RNAi technology to silence SARS-CoV S genes incultured cells.To develop new anti-SARS-CoV RNAi drug, we first establishedtwo-color fluorescence RNAi technique platform; and by using thisplatform, we tested whether shNP953, a self-designed novel shorthairpin RNA based on the sequence 953~971bp of nucleocapsid protein(NP), could knockdown gene expression of SARS-CoV NP. In addition,we studied RNAi mediated by U6-shRNA PCR Cassette.The main parts of our study are as follows:1 The establishment of RNAi technique platform aimed at studyingthe silencing of exogenous gene In order to establish a universal RNAi technique platform aimed atstudying the silencing of exogenous gene, we constructed two-colorfluorescence RNAi system in cis and trans patterns separately bysynthesizing small hairpin RNA under human U6 promoter in vivo.1.1 Construction of two-color fluorescence cis RNAi system and its application in RNAi study We constructed two coexpression vectors, named pcDNA3.0/GFP-U6-shGFP and pcDNA3.0/GFP-U6, which both encodeexogenous reporter gene (GFP). The difference lies between them iswhether containing GFP interfering RNA (shGFP) coding sequence.We also constructed a eukaryotic expression vector, namedpcDNA3.0/DsRed, which encodes red fluorescence protein (RFP)functioning as an indicator of transfection efficiency. These vectorsconstitute the two-color fluorescence cis RNAi system. ThepcDNA3.0/DsRed plasmid was co-transfected into Hela cells witheither the RNAi plasmid pcDNA3.0/GFP-U6-shGFP or control plasmidpcDNA3.0/GFP-U6. We then assayed green fluorescence intensitywithin the same number of transfected cells (indicated by RFP) usingflow cytometry. The results demonstrated that the two-color fluorescence RNAisystem constructed as cis pattern could not be used efficiently in RNAistudy.1.2 Construction of two-color fluorescence trans RNAi system and its application in RNAi study To deal with the problems, such as low RNAi efficacy, inability toshow shRNA transfection efficiency simultaneously and dose-dependent characteristic of RNAi, which were caused by two-colorfluorescence RNAi cis system, we developed the two colorfluorescence RNAi system in another way. This time we alsoconstructed two coexpression vectors, named pcDNA3.0/DsRed-U6-shGFP and pcDNA3.0/DsRed-U6, which substitute RFP gene for GFPgene. Together with the pcDNA3.0/GFP vector, which encode GFP, wegot the two color fluorescence trans RNAi system. Either the pcDNA3.0/DsRed-U6-shGFP or pcDNA3.0/DsRed-U6vector was cotransfected with the pcDNA3.0/GFP plasmid into Helacells. To assess the RNAi efficacy, we selectively analyze greenfluorescence intensity at the same red fluorescence background by flowcytometry and fluorescence microscopy quantitatively and qualitatively.We also tested the level of GFP mRNA by semi-quantificationRT-PCR. The results demonstrated GFP can be effectively and specificallysuppressed at both protein and mRNA level using this two-colorfluorescence trans RNAi system. This system not only has theadvantage of direct detection of target gene knockdown through greenfluorescence and monitoring interfering RNA transfection efficiencythrough red fluorescence, but also enriches the transfection efficiencyrelatively by selectively analyzing the GFP level of red fluorescencecells. Thus, we established a universal RNAi technique platform whichprovides feasibility to further study RNAi of SARS-CoV NP gene2. Application of two-color fluorescence RNAi technique platform in the study of SARS-CoV NP silencing We tried to study SARS-CoV NP RNAi using the establishedtwo-color fluorescence trans RNAi technique platform. According to the construction pattern of established two-colorRNAi trans system, we subcloned the full length coding gene ofSARS-CoV NP in-frame into the downstream of GFP gene to createGFP-NP fusion gene (pcDNA3.0/GFP-NP); then substitute shNP953 (aself-designed novel short hairpin RNA based on the sequence953~971bp of NP) for shGFP to construct coexpression vector namedpcDNA3.0/DsRed-U6-shNP953. Thus we got the two-colorfluorescence trans-RNAi system aimed at study of SARS-CoV RNAi.We carried out RNAi study on SARS-CoV NP by co-transfecting thesetwo vectors into Hela cells. The results demonstrated that SARS-CoV NP could besuccessfully suppressed by shNP953 using this two-color fluorescencetrans RNAi platform.3. The effect of U6-shRNA PCR Cassettes on SARS-CoV NP RNAi and induction of anti-virus substances Several groups have demonstrated that the shRNA expressioncassette generated by PCR method can not only effectively knockdowntarget gene expression, but also provide a rapid, easy and cheapapproach for screening candidate siRNA sequences. So, we studiedRNAi using U6-shRNA PCR Cassette.3.1 U6-shRNA PCR Cassette mediating SARS-CoV NP RNAi U6-shRNA PCR Cassettes contain the human U6 promoter andthe coding sequences for shRNA, followed by six Ts as polymerase â…¢RNA polymerase terminator. We got altogether five differentU6-shRNA PCR Cassettes and co-transfected each of these Cassettestogether with pcDNA3.0/GFP-NP and pcDNA3.0/DsRed into Helacells to study SARS-CoV RNAi. The results demonstrated that U6-shGFP PCR Cassette candown-regulate GFP-NP significantly, whereas U6-shNP570 PCRCassette and U6-shNP669 PCR Cassette had only minimal effect,...