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Inhibition of hepatitis B virus gene expression and replication by artificial microrna

World Journal of Gastroenterology 2008年第29期14卷 4684-4689页

作者： Yu-Feng Gao Li Yu Wei Wei Jia-Bin Li Qing-Li Luo Ji-Long Shen Institute of Clinical Pharmacology Anhui Medical University Hefei 230032 Anhui Province China Anhui Key Laboratory ofZoonoses Hefei 230032 Anhui Province China The Key Laboratory of Gene Resource Utilization for Severe Diseases The Ministry of Education of China and Anhui Province Hefei 230032 Anhui Province China Department of Infectious Diseases The First Affiliated Hospital of Anhui Medical University Hcfci 230032 Anhui Province China

AIM: To investigate the inhibitory effects of hepatitis B virus (HBV) replication and expression by transfecting artificial microrna (amiRNA) into HepG2.2.15 cells. METHODS: Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays (TRFIA). HBV DNA replication was examined by ? uorescence quantitative PCR, and the level of HBV S mRNA was measured by semi- quantitative RT-PCR. RESULTS: The efficiency of transient transfection of the vectors into 2.2.15 cells was 55%-60%. All the vectors had significant inhibition effects on HBsAg and HBeAg at 72 h and 96 h after transfection (P microrna targeting the HBV S coding region. Vector-based artificial microrna could be a promising therapeutic approach for chronic HBV infection.

关键词： Hepatitis B virus RNA interference artificial microrna HepG2.2.15 cell

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Inhibition of BmNPV replication in silkworm cells using inducible and regulated artificial microrna precursors targeting the essential viral gene lef-11

Inhibition of BmNPV replication in silkworm cells using indu...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Jun Zhang Qian He Chun-Dong Zhang Xiang-Yun Chen Xue-Mei Chen Zhan-Qi Dong Na Li Xiu-Xiu Kuang Ming-Ya Cao Cheng Lu Min-Hui Pan State Key Laboratory of Silkworm Genome Biology Southwest University Department of Biochemistry and Molecular Biology Chongqing Medical University Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry Southwest University

Bombyx mori nucleopolyhedrovirus（Bm NPV） is a major silkworm pathogen, causing substantial economic losses to the sericulture industry annually. We demonstrate a novel anti-Bm NPV system expressing mature artificial micro RNAs（ami RNAs） targeting the viral lef-11 gene. The mature amiRNAs inhibited the lef-11 gene in silkworm BmN-SWU1 cells. Antiviral assays demonstrated that mature ami RNAs silenced the gene and inhibited BmNPV proliferation efficiently. As constitutive overexpression of mature ami RNAs may induce acute cellular toxicity, we further developed a novel virus-induced ami RNA expression system. The amiRNA cassette is regulated by a baculovirus-induced fusion promoter. This baculovirus-induced RNA interference system is strictly regulated by virus infection, which functions in a negative feedback loop to activate the expression of mature amiRNAs against lef-11 and subsequently control inhibition of BmNPV replication. Our study advances the use of a regulatable amiRNA cassette as a safe and effective tool for research of basic insect biology and antiviral application.

关键词： BmNPV RNA interference artificial microrna Baculovirus-induced RNA interference system

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Efficient Silencing of Endogenous micrornas Using artificial micrornas in Arabidopsis thaliana

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Molecular Plant 2011年第1期4卷 157-170页

作者： Andrew L. Eamens Claire Agius Nell A. Smith Peter M. Waterhouse Ming-Bo Wang CSIRO Plant Industry GPO Box 1600 Canberra ACT 2601 Australia School of Molecular Bioscience University of Sydney Sydney NSW 2006 Australia

We report here that the expression of endogenous micrornas （miRNAs） can be efficiently silenced in Arabi- dopsis thaliana （Arabidopsis） using artificial miRNA （amiRNA） technology. We demonstrate that an amiRNA designed to target a mature miRNA directs silencing against all miRNA family members, whereas an amiRNA designed to target the stem-loop region of a miRNA precursor transcript directs silencing against only the individual family member targeted. Furthermore, our results indicate that amiRNAs targeting both the mature miRNA and stem-loop sequence direct RNA silencing through cleavage of the miRNA precursor transcript, which presumably occurs in the nucleus of a plant cell during the initial stages of miRNA biogenesis. This suggests that small RNA （sRNA）-guided RNA cleavage in plants occurs not only in the cytoplasm, but also in the nucleus. Many plant miRNA gene families have been identified via sequencing and bio- informatic analysis, but, to date, only a small tranche of these have been functionally characterized due to a lack of ef- fective forward or reverse genetic tools. Our findings therefore provide a new and powerful reverse-genetic tool for the analysis of miRNA function in plants.

关键词： microrna artificial microrna RNA silencing.

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Dimeric artificial micrornas mediate highly efficient RSV and RBSDV resistance in transgenic rice plants

Dimeric artificial microRNAs mediate highly efficient RSV an...

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中国植物病理学会第十二届青年学术研讨会

作者： Lin Sun Chao Lin Yunzhi Song Jinwen Du Fujiang Wen Changxiang Zhu State Key Laboratory of Crop Biology Shandong Key Laboratory of Crop BiologyCollege of Life SciencesShandong Agricultural University

Infection of Rice stripe virus（RSV） and Rice black streaked dwarf virus（RBSDV） on rice results in severe economic losses on crop *** the natural resistance resources against these viruses are limited,it is imperative to elaborate a biotechnological approach that will provide effective and safe immunity to RSV and *** this study,we generated three dimeric amiRNA precursor expression vectors（pamiR-M,pamiR-3 and pamiR-U） that simultaneously targeting RSV and RBSDV based on the structure of rice osa-MIR528 *** transgenic rice plants were obtained by Agrobacterium tumefaciens-mediated transformation and proved to express amiRNAs *** viral challenge assays revealed that these transgenic plants could be conferred highly specific resistance against RSV and RBSDV infection *** amiR-RSVU and the amiR-RBSDVU induced the highest virus resistance against RSV and RBSDV,*** target viral RNAs could be cleavaged by the amiRNAs and there was a relationship between the resistance level and the accumulation of amiRNA expression.A Northern blot assay verified that silencing was induced by the original amiRNAs and could be bilaterally extended by the siRNA *** amiRNA together with the secondary siRNAs mediated the degradation of viral *** stability assay showed that transgene and amiRNAmediated virus resistance can be stably inherited toT2.

关键词： artificial microrna Rice stripe virus Rice black streaked dwarf virus multiple virus resistances transgenic rice

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Identification of Male sterility-related Genes in Brassica napus toward a novel design for both pollination control system and disease control

Identification of Male sterility-related Genes in Brassica n...

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湖北省遗传学会第八次代表大会暨学术讨论会

作者： Wang Zhuoyu,Dong Caihua,Liu shengyi~* Oil Crops Research Institute,Chinese Academy of Agricultural Sciences,Wuhan,430062, China

Male sterility is an inheritable trait characterized by the inability of a plant to produce functional *** main types of male sterility in plant were discovered:cytoplasmic male sterility(CMS) and genic male sterility(GMS).GMS is a type of male sterility controlled by nuclear *** male sterility genes may play roles in different development stages of microspore *** on the expression profile analysis of Brassica napus male sterility material Shaan-GMS using Arabidopsis thaliana ATH-1 microarray,some greatly diversified expressing genes were selected to identify and investigate the effect of those genes on genetic male *** this purpose,artificial microrna vectors were used to interfere expression of these genes in *** target sequences of each gene were designed for microrna *** vectors were transferred into Arabidopsis by a method of flower *** transgenic Arabidopsis was screened with 5ppm *** phenotypes of transgenic offspring were observed during microspore *** and cellular methods were used to further identify functions of Ms-related *** methods included PCR for screening out the transgenic Arabidopsis,Q-PCR for validating microrna vector’s interfering efficiency, Arekisanda staining for identification of the extent of microspore *** them,some genes have been identified involving in pollen *** positively transgenic plants had no pollen during the flowing period and showed microspore abortion by Alexander staining.Q-PCR validated that expression levels of the gene in these transgenic lines are about 1-5%of the wild *** results showed that interfering by artificial microrna leads to post-transcriptional gene silencing,which caused male *** characterization of the gene can contribute to the understanding of the molecular mechanisms of fertility control and stamen development in oilseed *** these results, we have developed a wor

关键词： DGMS artificial microrna realtime PCR

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