Stable plasmid-based siRNA silencing of gene expression in human embryonic stem cells

RNA interference (RNAi) using short inhibitory RNAs (siRNAs) has been widely explored for the suppression of cellular mRNA levels to investigate the function of specific genes, including gene function in differentiation and development. The establishment of human embryonic stem cell (hESC) models for differentiation of selected lineages is an area of intense interest and activity. On the basis of our previous work with stable overexpression of enhanced green fluorescent protein (EGFP) in hESC, we used plasmid vector-based siRNA expression to silence EGFP expression in stably-transfected hESC. After hygromycin selection, we derived several cell lines in which EGFP expression was significantly reduced. At the genomic DNA level, there was no difference between the two cell lines and the parental H1EGFP cell line when analyzed with quantitative PCR; however, there were significant differences among the three cell lines at the RNA and protein levels as analyzed with real-time RT-PCR and Western blotting. From these data, we conclude that the decrease in EGFP expression was caused by RNAi, not by genomic DNA loss. Down-regulation of EGFP expression was sustained through multiple passages of both siEGFP cell lines. This simple silencing system will allow novel investigations of target gene function in hESC self-renewal or differentiation, as well as differentiated function in other cell types.     

Silencing structural and nonstructural genes in baculovirus by RNA interference

We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.     

Reconstitution of TCP80/NF90 translation inhibition activity in insect cells

Acid beta-glucosidase (GCase) is the enzyme deficient in Gaucher disease, an inherited metabolic prototype for enzyme and gene therapy. An 80-kDa mammalian cytoplasmic protein (TCP80/NF90) was discovered to interact with the GCase mRNA coding region and inhibit its translation in vitro and ex vivo. Human TCP80/NF90 is identical to NF90, an IL-2 enhancer protein, and MPP4, an M-phase phosphoprotein. The interaction of recombinant TCP80/NF90 with GCase mRNA was evaluated using the baculovirus/Sf9 insect cell system since these cells lack this protein. Purified recombinant and isolated mammalian cytoplasmic TCP80/NF90 had identical functions including binding of coding regions of selected RNAs and inhibition of their in vitro translation. Individual baculoviruses containing the human TCP80/NF90 cDNA (vSf9/TCP80) and GCase cDNA (vSf9/GCase) were used to co-infect Sf9 cells. The presence of preformed TCP80/NF90 significantly (>87%) inhibited wild-type GCase mRNA translation in these cells, but baculovirus containing a mutant GCase did not. Sf9 cells co-infected with vSf9/TCP80 showed a major reduction of GCase RNA polysome association. These results show that the multifunctional protein, TCP80/NF90, can function in vivo as a translation inhibitory protein and include alterations of mRNA binding to polysomes as a component of its mechanism of action.     

P1 Protein of Cocksfoot Mottle Virus is Indispensable for the Systemic Spread of the Virus

Cocksfoot mottle sobemovirus (CfMV) encodes a non-conserved protein P1 from the 5′ ORF1 of genomic RNA. The functions of CfMV P1 are unknown. In the current study we show that P1-deficient CfMV can replicate both in oat leaves and barley suspension culture cells but can not infect oat plants systemically. However, the absence of P1 reduces the efficiency of virus accumulation considerably. The infectivity of the mutant virus restores as a result of the spontaneous transversion. CfMV P1:EGFP shows a very limited cell-to-cell movement in leaf epidermal cells. In Sf9 insect cells CfMV P1 localizes in the fraction of membranes and inclusions but not in soluble cytoplasmic protein fraction.     

基因沉默PRDX1 表达对人结直肠癌SW480 细胞侵袭迁移的影响

目的:探讨RNA 干扰过氧化还原酶1(Peroxiredoxin 1,PRDX1)表达对人结直肠癌SW480 细胞侵袭转移能力的影响。方法:筛选RNA 干扰PRDX1 的慢病毒质粒,与阴性对照慢病毒质粒分组转染结直肠癌SW480 细胞,转染后的SW480 细胞可分为PRDX1 基因沉默组(si-PRDX1)和阴性对照组(Vector)。实时荧光定量PCR(qRT-PCR)和免疫印迹法(Western blot)分别检测两组细胞中PRDX1 mRNA 和蛋白表达;采用Transwell 侵袭和迁移实验检测基因沉默PRDX1 表达对结直肠癌细胞侵袭及迁移能力的影响;通过Western blot 检测两组细胞中基质金属蛋白酶(MMP)家族部分蛋白表达水平。结果:基因沉默PRDX1 表达可有效抑制结直肠癌SW480 细胞中PRDX1 mRNA 和蛋白水平的表达,与阴性对照组相比(Vector),差异均具有统计学意义(P<0.01),说明基因沉默PRDX1 的SW480 细胞系构建成功;Transwell 侵袭和迁移实验显示si-PRDX1组细胞的侵袭及迁移能力较对照组均明显降低(P<0.01);Western blot 结果显示,与Vector 组相比,si-PRDX1 组细胞中组织基质金属蛋白酶抑制剂2(TIMP-2)的表达明显增加,而MMP-2 及MMP-9 的表达显著下降,且差异均具有统计学意义(P<0.05)。结论:基因沉默人结直肠癌SW480 细胞的PRDX1 表达可有效抑制细胞的侵袭、迁移及转移能力,其机制可能会通过调控TIMP-2、MMP-2 及MMP-9 的表达介导。     

The development of vector constructions for respiratory syncitial virus (RSV) P-gene silencing

Interfering RNA (RNAi) is a powerful tool to silence gene expression on the level of mRNA. To knock-down gene expression by using RNAi two major methods of mRNA silencing exist. First method utilizes siRNA (small interfering RNA), a readily processed dsRNA, that enters RISC complex and destroy target mRNA after transfection into the cells. The second method based on the construction of plasmid DNA that expresses shRNA (short harpin RNA) from U6 or CMV promoter. shRNA gets processed by Drosha and Dicer RNAses inside the cell before it translocates to the cell cytoplasm and affects the level of target RNA. In this study we modified lentiviral vector pGIPZ expressing tFP-IRES-Puro-shRNA(mir30) cassette by introducing BamH I restriction site downstream of this cassette. This modification makes possible to clone specific shRNA sequences in pGIPZ vector using XhoI/BamHI restriction sites instead of the original recombination. Three shRNAs against phosphoprotein P of respiratory sinthitial virus (RSV) and shRNA against human CD43 as a control were generated and cloned into modified so-called pCIPD vector. Monkey kidney cells MA-104 were stably transduced with four shRNA constructs. In conclusion, the generated lentiviral vector pCIPD can be successfully used for efficient gene silencing and virus replication in a broad variety of cells.     

Interfering with hepatitis C virus RNA replication

The emergence of RNA interference (RNAi) as a powerful tool for silencing gene expression has spurred considerable interest in its experimental and therapeutic potential. RNAi is a cellular process of gene silencing in which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. The introduction of 21-23 nucleotide RNA duplexes, termed small interfering RNAs (siRNAs), into mammalian cells can specifically degrade homologous mRNAs. RNAi efficiently silences the expression of both cellular and viral RNAs. A number of groups have demonstrated that siRNAs interfere with hepatitis C virus (HCV) gene expression and replication. Additionally, cellular genes are efficiently silenced in the presence of replicating HCV. These studies lay the foundation for using RNAi as an experimental tool for studying HCV replication and defining host genes that are significant for viral replication. The potential for RNAi as an antiviral therapy remains less clear, as it will face many of the challenges that have hindered nucleic acid therapies in the past.     

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins

Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease.     

大鼠β防御素2基因RNAi慢病毒载体的构建及效应测定

目的 构建大鼠β防御素2(rBD2)基因RNAi慢病毒重组载体,转染培养细胞,检测其沉默效应,筛选出最佳的RNAi慢病毒载体.方法 序列软件设计3条针对rBD2基因CDS区的siRNA序列,合成单链后退火形成双链DNA,分别与酶切处理的慢病毒载体lentivirus连接构成3个RNAi慢病毒重组载体,再转化细菌,测序鉴定.脂质体法转染细胞,荧光实时定量PCR(RT-PCR)及Western免疫印迹测rBD-2 mRNA及蛋白表达,筛选沉默效果最佳的为LV-shrBD2载体.用慢病毒包装系统对LV-shrBD2进行慢病毒颗粒包装并梯度稀释法测定病毒滴度.结果 凝胶电泳显示3个RNAi慢病毒重组载体的PCR产物为316 bp,测序结果表明序列正确.转染细胞后RT-PCR及Western免疫印迹检测,siRNA序列1构建的重组载体mRNA抑制率达82%,干扰效率最高,为所需的rBD2基因RNAi慢病毒载体LV-shrBD2.包装慢病毒颗粒并调整病毒滴度至1×105ifu/μl.结论 成功构建并筛选出沉默效应最佳的rBD2基因RNAi慢病毒表达载体LV-sh1rBD2,为进一步开展rBD2研究提供了依据.     

含人过氧化物酶体增殖物激活受体δ基因高效真核表达载体的构建及其意义

目的 构建高效表达人过氧化物酶体增殖物激活受体δ(hPPARδ)的真核表达载体,为hPPARδ受体功能和基于hPPARδ受体靶点的药物筛选提供分子研究平台.方法 采用逆转录-聚合酶链式反应(RT-PCR),从HepG2细胞总RNA克隆hPPARδ全长基因,与经BamHI、SalI相同双酶切的pIRES2-EGFP载体连接,构建重组质粒phPPARδ-IRES2-EGFP,经酶切及基因测序鉴定重组质粒中hPPARδ基因的完整性和忠实性;荧光显微镜观察重组质粒转染的293细胞GFP报告基因表达强度,并对转染细胞hPPARδ的表达进行荧光定量PCR和免疫细胞化学检测.结果 经酶切和测序证实重组质粒构建正确,并在转染的293细胞中获得hPPAR6的高效表达.结论 成功构建phPPARδ-IRES2-EGFP重组质粒.