利用人工microRNA实现基因沉默

MicroRNA是一组长度约为21 nt的非编码蛋白质的短序列RNA,能通过碱基互补配对的方式指导降解靶基因mRNA或抑制靶基因的翻译。MicroRNA的主要功能是调控基因的表达,在生物体的生长、发育及疾病发生中扮演着重要的角色。本文介绍了利用microRNA实现基因沉默的作用原理,人工合成microRNA,构建转基因载体,实现对目的基因的沉默及这种工具在生命科学领域的应用前景。     

p53 Gene Targeting by Homologous Recombination in Fish ES Cells

Background

Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes).

Methodology and Principal Findings

Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1∼MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by ∼12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo.

Conclusions

Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.     

amiRNAi-实现高效稳定的特异基因沉默新方法

RNA干扰技术(RNA interference,RNAi)是实现基因沉默的有效工具。近年来,随着分子生物学与生物技术的快速发展,在RNAi基础上又发展出另一种特异性更高的基因沉默技术—amiRNAi (artificial microRNA interference)。amiRNAs是一类由内源miRNA前体生成的长21个核苷酸的人工小RNA分子,它能在不影响其他基因表达的情况下特异地介导单个或多个靶基因高效稳定沉默。与普通的RNAi相比,amiRNAi具有特异性高、稳定性强和沉默效应可预见等优点。因而amiRNAi可能成为基因功能分析的最有效工具之一,同时amiRNAi对于基因负调控研究和应用而言前景广阔。着重介绍了amiRNAi技术的原理、优势及其潜在的应用价值。     

Specific Inhibition of Tumor Cells by Oncogenic EGFR Specific Silencing by RNA interference

Anticancer agents that have minimal effects on normal cells and tissues are ideal cancer drugs. Here, we show specific inhibition of human cancer cells carrying oncogenic mutations in the epidermal growth factor receptor (EGFR) gene by means of oncogenic allele-specific RNA interference (RNAi), both in vivo and in vitro. The allele-specific RNAi (ASP-RNAi) treatment did not affect normal cells or tissues that had no target oncogenic allele, whereas the suppression of a normal EGFR allele by a conventional in vivo RNAi caused adverse effects, i.e., normal EGFR is vital. Taken together, our current findings suggest that specific inhibition of oncogenic EGFR alleles without affecting the normal EGFR allele may provide a safe treatment approach for cancer patients and that ASP-RNAi treatment may be capable of becoming a safe and effective, anticancer treatment method.     

转录后基因沉默与植物的病毒抗性

转录后基因沉默(PTGS)是近10年发现的一种生物(特别是真核生物)细胞抵抗外来核酸入侵及保持生物自身基因组完整性的防御机制,特别是与生物的病毒抗性密切相关。PTGS最初在植物内发现,近几年又分别在真菌、动物等生物细胞内发现。经过10年的研究,我们对PTGS的机制和特点有了相当的了解。这不但对深入地了解基因的表达调控机制意义重大,而且还可为人们如何调控和利用PTGS奠定了基础。本文从PTGS的特点、PTGS与病毒抗性、PTGS在真核生物内发生的广泛性等方面进行综述,并对PTGS发生的机制进行了讨论。     

逆转录病毒载体介导的猪瘟病毒配基因的真核表达

利用DNA重组技术将猪瘟病毒(Classicalswinefevervirus,CSFV)石门株囊膜蛋白E2基因插入逆转录病毒载体pBABE-puro中构建成重组逆转录病毒载体pBABE-puro-E2,该重组逆转录病毒载体与pVSVg质粒经磷酸钙共转染法将其转入293GP细胞中包装逆转录病毒假病毒。用包装的假病毒感染PK-15细胞,经嘌呤霉素筛选阳性细胞后进行流式细胞技术(FACS)分析,结果表明CSFVE2基因在PK-15细胞膜上成功表达。将表达E2蛋白的PK-15细胞腹腔免疫Balb/c小鼠,成功诱导小鼠产生了抗E2蛋白的抗体。     

逆转录病毒载体介导的猪瘟病毒E2基因的真核表达

利用DNA重组技术将猪瘟病毒(Classical swine fever virus , CSFV)石门株囊膜蛋白E2基因插入逆转录病毒载体pBABE-puro 中构建成重组逆转录病毒载体pBABE-puro-E2,该重组逆转录病毒载体与pVSVg质粒经磷酸钙共转染法将其转入293GP细胞中包装逆转录病毒假病毒.用包装的假病毒感染PK-15细胞,经嘌呤霉素筛选阳性细胞后进行流式细胞技术(FACS)分析,结果表明CSFV E2基因在PK-15细胞膜上成功表达.将表达E2蛋白的PK-15细胞腹腔免疫Balb/c小鼠,成功诱导小鼠产生了抗E2蛋白的抗体.     

Silencing of HIV-1 Gene Expression by Sirnas in Transduced Cells

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (≥90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490–7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.     

Pten基因沉默可抑制子宫内膜细胞增殖

本研究将Pten-siRNA转染至子宫内膜细胞,通过蛋白免疫印迹检测子宫内膜细胞中Pten蛋白的表达情况;通过MTT法检测Pten-siRNA转染组和空质粒转染组(对照组)细胞增殖状态;通过蛋白免疫印迹检测Pten-siRNA转染组和对照组子宫内膜细胞中PCNA、Ki67蛋白表达情况;通过流式细胞仪检测子宫内膜细胞Pten-siRNA转染组和对照组中ROS的水平,以探究Pten基因对子宫内膜癌发生发展的影响。研究表明,Pten-siRNA转染后,子宫内膜细胞中Pten蛋白明显低于对照组;Pten-siRNA转染后,子宫内膜细胞增殖率显著高于对照组(p<0.05);Pten-siRNA转染后,细胞增殖相关蛋白PCNA和Ki67明显高于对照组(p<0.05);Pten-siRNA转染后,子宫内膜细胞中ROS水平明显低于对照组(p<0.05)。本研究初步结论显示Pten-siRNA可降低子宫内膜细胞中Pten蛋白表达,抑制子宫内膜细胞的增殖并增加子宫内膜细胞中ROS水平。     

Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

Virus-induced gene silencing （VIGS） is a useful technique for rapid plant gene function analysis. We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus （TYLCCNV） DNAβ （DNAm β）. In this study we compared in detail DNAmβ-induced gene silencing in four Nicotiana species including N. benthamiana, N. glutinosa, N. tabacum and N. paniculata. We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span. It was most efficient in N. benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient. DNAmβ-induced gene silencing in N. glutinosa was also efficient despite being slightly less than in N. benthamiana. It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves. In both species, VIGS constantly expressed until the plants died. However, DNAmβ-mediated VIGS in the other two Nicotiana species, N. tabacum and N. paniculata, was significantly less efficient. It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks. The upper leaves that emerged later stopped showing the silencing phenotype. DNAmβ-induced gene silencing in N. benthamiana and N. glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated, and was not sensitive to high growth temperature up to 32℃. Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.     

病毒诱导的PVX cp转基因沉默及其DNA甲基化

利用PCR方法获得了马铃薯X病毒(PVX)外壳蛋白(CP)基因(cp),并将其构建到植物表达载体中,利用农杆菌介导的叶盘法转化烟草(Nicotiana tabacum L.).Northern杂交及Run on实验表明有3株转基因烟草发生了转录后基因沉默.发生沉默的cp基因的甲基化分析结果表明,发生转录后基因沉默的cp基因发生了不同程度的甲基化,说明DNA甲基化并没有完全抑制cp基因的转录.利用PVX病毒对外壳蛋白正常表达的转基因烟草进行接毒,Northern杂交检测结果表明,病毒诱导cp发生了基因沉默.进一步的Run on结果表明,转基因烟草中cp基因在沉默前后转录速率并没有发生变化,说明病毒诱导的沉默是一种转录后沉默.对cp基因沉默前后的甲基化分析表明,病毒的侵染导致了cp基因甲基化程度的增加.     

Double-stranded RNA Mediates Selective Gene Silencing of Protein Phosphatase Type 1 Delta Isoform in HEK-293 Cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1δ) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP18. This RNA interference did not affect the expression of α and γ1 isoforms of PP1. Transfection of antisense RNA specific for PP1δ also suppressed the expression of PP1δ. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.     

Gene Silencing Mediated by Endogenous MicroRNAs under Heat Stress Conditions in Mammalian Cells

Heat shock, sudden change in temperature, triggers various responses in cells for protecting the cells from such a severe circumstance. Here we investigated gene silencing mediated by endogenous microRNAs (miRNAs) in mammalian cells exposed to a mild hyperthermia, by means of miRNA activity assay using a luciferase reporter gene as well as miRNA expression analysis using a DNA microarray. Our findings indicated that the gene silencing activities involving miRNAs were enhanced without increasing in their expression levels under heat-stress conditions. Additionally, the gene silencing activity appeared to be independent of the cytoprotective action involving heat shock proteins that are immediately activated in heat-shocked cells and that function as molecular chaperons for restoring heat-denatured proteins to normal proteins. Our current findings suggested the possibility that gene silencing involving endogenous miRNAs might play a subsidiary role in heat-shocked cells for an aggressive inhibition of the expression of heat-denatured proteins.     

Enhanced Gene Silencing in Cells Cured of Persistent Virus Infection by RNA Interference

We compared HEp-2-derived cells cured of persistent poliovirus infection by RNA interference (RNAi) with parental cells, to investigate possible changes in the efficiency of RNAi. Lower levels of poliovirus replication were observed in cured cells, possibly facilitating virus silencing by antiviral small interfering RNAs (siRNAs). However, green fluorescent protein (GFP) produced from a measles virus vector and also GFP and luciferase produced from plasmids that do not replicate in human cells were more effectively silenced by specific siRNAs in cured than in control cells. Thus, cells displaying enhanced silencing were selected during curing by RNAi. Our results strongly suggest that the RNAi machinery of cured cells is more efficient than that of parental cells.Small interfering RNAs (siRNAs) mediate RNA interference (RNAi), a natural biological phenomenon regulating a wide range of cellular pathways (8, 20). RNAi-based therapies with siRNAs or small hairpin RNAs (shRNAs) have been developed against several viral infections, and a reduction of the viral yield by several orders of magnitude has frequently been obtained (4, 9). However, virus clearance from cells and the complete cure of persistent virus infections have only rarely been reported (24, 25). We have developed several models of persistent virus infection by using poliovirus (PV), a positive-strand RNA virus of the Picornaviridae family (5, 7, 16, 21). We previously studied the effects of antiviral siRNAs applied months after the infection of HEp-2 cells with a persistent PV mutant (7, 25). We used a mixture (“the Mix”) of two synthetic siRNAs targeting the viral RNA genome in the 5′ noncoding (NC) region and the 3D polymerase (3D^pol) (siRNA-5′NC and siRNA-3D^pol, respectively; synthesized by Sigma-Proligo). When repeated transfections with the Mix were performed in persistently PV-infected cultures, most cultures stopped producing virus (25). Here, we investigate the important issue of changes in RNAi efficacy following siRNA treatment, 2 to 5 months after the cure. The efficiency of gene silencing in cells was stable during this period.We used the HEp-Q4 and -Q5 cell lines, which were cured of persistent PV infection after transfections with the Mix (25). The cured cells and their parental cell line, HEp-2, had similar growth rates (data not shown). To compare PV silencing efficiencies in the three cell lines, they were transfected either with the Mix or with an irrelevant siRNA (siRNA-IRR) in the presence of Lipofectamine 2000 (Invitrogen) in 24-well plates as previously described (25). Treated and mock-treated cells were infected 16 h posttransfection with PV strain Sabin 3, at a multiplicity of infection (MOI) of 1 50% infectious dose (ID₅₀) per cell. The viral progeny was titrated 24 h postinfection, as previously described (16). HEp-Q4 and HEp-Q5 were permissive to PV infection, although viral yields were about 1 log lower in these cells than in HEp-2 cells (Fig. ​(Fig.1A).1A). Virus silencing was observed in all three cell lines treated with the Mix; however, silencing was significantly more efficient in HEp-Q4 (≈2.2 times more efficient; P = 0.013, Student''s t test) and HEp-Q5 (≈5.6 times more efficient; P = 0.015) than in HEp-2 cells (Fig. 1A and B). Similar results were obtained with an shRNA (Thermo Scientific) targeting the same region as the siRNA-5′NC (data not shown).Open in a separate windowFIG. 1.Efficiency of enterovirus silencing in HEp-2, HEp-Q4, and HEp-Q5 cells after transfection with specific siRNAs. (A) Yield of progeny virus produced by cells infected at an MOI of 1 ID₅₀, 16 h posttransfection with the antiviral Mix containing two anti-PV siRNAs (20 pmol), the irrelevant siRNA-IRR (20 pmol), or no siRNA. Samples were harvested 24 h postinfection. Each bar represents the mean value ± SEM of six infected cultures from three independent experiments. (B to E) For each cell line, silencing efficiency is expressed as the ratio of infectious virus yield (titer in ID₅₀/ml) in the presence of the irrelevant siRNA-IRR to infectious virus yield (titer in ID₅₀/ml) in the presence of the antiviral siRNAs in cured cells, normalized with respect to the silencing efficiency in HEp-2 cells. S2, PV strain Sabin 2. (F) GFP silencing efficiency for each cell line is expressed as a ratio [1 − (mean GFP levels in the presence of siRNA-eGFP)/(mean GFP levels in the presence of siRNA-IRR)] in cured cells, normalized with respect to the efficiency of silencing in HEp-2 cells. Each bar represents the mean value ± SEM of at least four cultures from two independent experiments. *, P < 0.05 based on Student''s t test comparing HEp-Q4 and HEp-Q5 with HEp-2 cells.We investigated whether the differences in silencing efficacies between the three cell lines were due to differences in siRNA transfection efficiency by transfecting HEp-2, HEp-Q4, and HEp-Q5 cells with fluorescein isothiocyanate-conjugated siRNA (siRNA-FITC; 20 pmol/well; Cell Signaling) and testing them between 4 and 48 h posttransfection. The fluorescence of transfected cells was measured with a FACScan flow cytometer (Becton Dickinson), and data were analyzed with CellQuest software (Becton Dickinson). The percentages of siRNA-FITC-positive cells were similar for all cell types (Fig. ​(Fig.2A).2A). The mean fluorescence per positive cell and the percentage of cells displaying fluorescence peaked 16 and 24 h posttransfection, respectively, and decreased thereafter (Fig. ​(Fig.2).2). These findings suggest both that the presence of siRNAs in cells was similarly transient in the three cell types, as previously reported (27), and that the high silencing efficiencies in cured cells were not a consequence of higher transfection efficiencies. All subsequent experiments were performed between 16 and 40 h posttransfection.Open in a separate windowFIG. 2.Transfection efficiencies of fluorescein-conjugated siRNAs in HEp-2, HEp-Q4, and HEp-Q5 cells. A fluorescent siRNA-FITC (20 pmol) was used to transfect each of the three cell lines in the presence of Lipofectamine 2000. Fluorescent cells were analyzed 4 to 48 h posttransfection by using a FACScan flow cytometer (Becton Dickinson). The percentage of fluorescent cells (A) and the mean fluorescence per positive cell, in arbitrary units (B), are shown. Each bar represents the mean value ± SEM. (C) Representative FACS plots (cell granularity versus cell size), showing the similarities between the three cell populations.Fluorescence-activated cell sorting (FACS) plots for granularity versus cell size were very similar for the three cell lines (Fig. ​(Fig.2C),2C), as were those for cell numbers versus fluorescence (not shown), suggesting highly related cell populations. Although highly probable, it remains to be confirmed that the cured cells originated from a subpopulation of HEp-2 cells.Virus silencing was also investigated in cured cells infected with Sabin 2 or coxsackievirus A17 (CAV17) strain 67591 (22) or in cells transfected with Sabin 2 RNA. The experimental conditions used for Sabin 2 and CAV17 were identical to those for Sabin 3, except that only the 3D polymerase was targeted by siRNAs. Sabin 2 RNA (1 μg) was prepared as previously described (12) and used with siRNA-3D^pol (20 pmol/well) for the cotransfection of cells in the presence of Lipofectamine 2000. Virus yields were determined 7.5 h after transfection. In all cases, virus silencing was more effective in HEp-Q4 and -Q5 cells than in HEp-2 cells (Fig. 1C to E). Additional experiments were performed with a PV replicon encoding the green fluorescent protein (GFP), PV-eGFP (28) (2 μg/well), which was used with siRNA-eGFP (20 pmol/well; Ambion) for cotransfection. GFP fluorescence was measured by flow cytometry, 16 h after transfection. As for PV, a higher silencing efficiency was observed in cured cells than in HEp-2 cells (Fig. ​(Fig.1F1F).We then investigated whether the lower level of viral multiplication in HEp-Q4 and -Q5 cells in the absence of siRNAs involved an entry or postentry step. We quantified the expression of the PV receptor (CD155) at the surface of cells. We used flow cytometry after indirect immunofluorescence labeling with anti-CD155 antibodies, as previously described (16). More than 98.4% ± 2% (mean ± standard error of the mean [SEM]) of cured cells, like HEp-2 cells, tested positive for CD155 (data not shown). In the absence of siRNAs, a decrease in viral replication was also observed in HEp-Q4 and -Q5 cells infected with the Sabin 2 PV strain in cells, in which the early stages of the viral cycle were bypassed by transfection with Sabin 2 RNA, and in cells infected with the CAV17 virus, which uses a cell receptor other than CD155 (12) (data not shown). Together, these results suggest that PV multiplication is reduced at a postentry step, probably at replication, in cured cells.We investigated whether PV silencing was also enhanced in other HEp-derived cells in which Sabin 3 PV multiplication was reduced by using HEp-S31 (cl18) cells that had been cured of persistent PV infection by growth at a supraoptimal temperature rather than by RNAi (2). PV yield was ≈1.6 logs lower in HEp-S31 (cl18) cells than in HEp-2 cells (data not shown). Sabin 3 PV silencing in HEp-S31 (cl18) cells was 1.7 ± 0.9 times more effective (mean of six experiments) than that in HEp-2 cells (relative efficacy of 1) (data not shown), but this difference was not significant. However, these results do not exclude the possibility that reduced PV replication facilitates PV silencing by the Mix in cured cells. We therefore pursued our work with a different virus.We investigated whether the high silencing efficiency in HEp-Q4 and -Q5 cells was specific to enteroviruses by using a measles virus expressing GFP, MV-eGFP (26), and siRNA-eGFP to silence GFP expression. Cells were transfected with either siRNA-eGFP or siRNA-IRR, infected with MV-eGFP (1 ID₅₀ per cell, 16 h posttransfection), and the GFP silencing efficiency was determined 40 h posttransfection by flow cytometry. For each cell line, silencing efficiency was expressed as a percentage {[1 − (percentage of siRNA-eGFP-transfected cells expressing GFP)/(percentage of siRNA-IRR-transfected cells expressing GFP)] × 100}. GFP silencing was significantly stronger in HEp-Q4 cells (≈14%; P = 0.048) and HEp-Q5 cells (≈17%; P = 0.010) than in HEp-2 cells (Fig. ​(Fig.3A).3A). There was no significant difference in the silencing efficiency of GFP between HEp-Q4 and -Q5 cells (Fig. ​(Fig.3A).3A). The anti-PV Mix did not silence GFP expression (data not shown), indicating that the silencing of GFP was not due to anti-PV siRNAs persisting in cured cells months after the initial treatment.Open in a separate windowFIG. 3.Efficiency of GFP and luciferase silencing in HEp-2, HEp-Q4, and HEp-Q5 cells after transfection with specific siRNAs. (A and B) GFP silencing, expressed as a percentage calculated for each cell line as follows: {[1 − (GFP expression in the presence of siRNA-eGFP)/(GFP expression in the presence of the irrelevant siRNA-IRR)] × 100}. (A) Cells were infected 16 h posttransfection with a measles virus encoding eGFP (MV-eGFP [26]) at an MOI of 1 ID₅₀/cell, and fluorescent cells were analyzed 24 h after infection (40 h posttransfection). Each bar represents the mean value ± SEM of three independent experiments. (B) Cells were cotransfected with pEGFP-C1 and siRNA-eGFP or siRNA-IRR and analyzed 40 h later. Each bar represents the mean value ± SEM of four independent experiments. (C) Luciferase silencing efficiency for each cell line, expressed as the ratio of luciferase activity in the presence of the irrelevant siRNA-IRR to luciferase activity in the presence of the specific siRNAs in cured cells, normalized with respect to silencing efficiency in HEp-2 cells. Relative efficiencies are shown as in Fig. ​Fig.11 for luciferase, because the enzymatic reaction amplified the signal. Each bar represents the mean value ± SEM of triplicates from three independent experiments. *, P < 0.05 based on Student''s t test comparing HEp-Q4 and HEp-Q5 with HEp-2 cells.To test whether the high silencing efficiency in HEp-Q4 and -Q5 cells was dependent on viral infection, plasmid vectors pEGFP-C1 (Clontech Laboratories) and pRL-CMV (Promega) were used to generate GFP (6) and Renilla luciferase (18), respectively. These plasmids do not replicate in human cells. Cells (10⁶) were cotransfected with pEGFP-C1 (1 μg) and siRNAs (20 pmol) in the presence of Lipofectamine 2000, as recommended by the manufacturer. GFP fluorescence was analyzed by flow cytometry 40 h posttransfection. Silencing efficiencies were expressed as a percentage {[1 − (mean GFP levels in the presence of siRNA-eGFP)/(mean GFP levels in the presence of siRNA-IRR)] × 100)}. Mean silencing efficiency was significantly higher in HEp-Q4 (≈15%; P = 0.003) and HEp-Q5 (≈15%; P = 0.002) cells than in HEp-2 cells (Fig. ​(Fig.3B).3B). The efficiency with which the GFP encoded by pEGFP-C1 was silenced was similar in HEp-Q4 and -Q5 cells.The efficacy of siRNAs was then assessed with pRL-CMV, which encodes the Renilla luciferase and Silencer Renilla luciferase (AM4630; Ambion). Cells (10⁶) were cotransfected with the plasmid (100 ng) and either specific or irrelevant siRNA (7 pmol) in the presence of Lipofectamine 2000. Luciferase assays were performed with a Dual-Glo luciferase assay system (Promega), as recommended by the manufacturer at 40 h posttransfection, and luminescence was measured with a luminometer (Centro LB960; Berthold). The results of the sensitive luciferase assays confirmed that the relative efficiency of silencing was significantly higher in cured than in parental cells (Fig. ​(Fig.3C).3C). By contrast, results obtained in HEp-S31 (cl18) cells, cured without siRNAs, were not significantly different from those obtained in control HEp-2 cells (data not shown), strongly suggesting that the treatment of HEp-Q4 and -Q5 cells with specific siRNAs selected cells in which siRNAs mediated silencing more efficiently than in parental cells.The difference in silencing efficiency between cured and HEp-2 cells may be due to differences in the abundance and/or efficacy of cellular factors involved in gene silencing. Some major actors of the RNAi pathway, particularly those associated with the RNA-induced silencing complex (RISC), have been identified (3, 10, 13, 19). The active endonucleolytic core of the RISC includes the guide strand of the siRNA and a slicer protein called Argonaute 2 (Ago2) (17). We used Western blotting to study Ago-2 and other factors contributing to the function of RISC (3, 10, 11, 14, 19, 23): the endonuclease Dicer, the transactivation response RNA binding protein (TRBP), the protein activator of double-stranded RNA-dependent protein kinase (PACT), and the RNA helicase A (RHA) (Fig. ​(Fig.4).4). Exportin 5, which plays a role upstream from the dicing process in the export of small RNA precursors (29), was included as a control.Open in a separate windowFIG. 4.Comparative analysis of proteins involved in RNAi in HEp-2, HEp-Q4, and HEp-Q5 cell lines. Whole-cell lysates were tested for Exportin 5 (A), Dicer (B), Ago-2 (C), the helicase RHA (D), TRBP (E to H) and PACT (I) by Western blotting with the corresponding specific antibodies. Blots were subsequently stripped and reprobed with antiactin antibodies to confirm equal protein loading. (E and F) TRBP levels in HEp-Q4 and HEp-Q5 cells were determined by densitometry and are plotted in arbitrary units, as ratios relative to the level of actin and to the level of TRBP in HEp-2 cells. In panel F the symbols correspond to TRBP levels determined in nine different experiments. (G) TRBP levels in HEp-2 cells transfected with pcDNA-TRBP (14) and in cells cotransfected with pcDNA-TRBP and siRNA-TRBP. (H) TRBP levels were compared in human IMR5 cells, HEpS31 (cl18) cells previously cured of persistent PV infection by growth at a supraoptimal temperature, and the control HEp-2 cell line. TRBP/actin densitometry and PACT/actin densitometry results are indicated in arbitrary units in the histograms below the corresponding Western blot results shown in panels H and I.Proteins (30 to 50 μg) from each cell line were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10 to 20% Tricine gels; Invitrogen) and transferred to nitrocellulose membranes (Amersham Biosciences) as previously described (1). The membranes were incubated with one of the following primary antibodies (1): anti-Ago2 monoclonal antibody (MAb; Abcam), anti-RHA MAb (Abcam), and anti-TRBP2 MAb (Santa Cruz Biotechnology); rabbit antibodies against Dicer (Santa Cruz Biotechnology); anti-PACT MAb (Santa Cruz Biotechnology), and anti-Exportin 5 MAb (Abcam). The antiactin MAb (AC-40; Sigma-Aldrich) was used to check for equal protein loading. Membranes were then washed and treated with appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) for 2 h at room temperature. Protein bands were detected with an enhanced chemiluminescence detection kit (ECL⁺; Amersham Biosciences) and a G:box (Syngene).Exportin 5, Dicer, Ago-2, and RHA were similarly abundant in all three cell lines (Fig. 4A to D), suggesting that quantitative differences in protein levels were unlikely to be responsible for the enhanced silencing in HEp-Q4 and -Q5 cells. There was significantly more TRBP in HEp-Q4 (≈21%; P = 0.026) and HEp-Q5 (≈28%; P = 0.016) cells than in HEp-2 cells, as indicated by the results of nine experiments (Fig. 4E and F). The specificity of the anti-TRBP antibody was checked on extracts of HEp-2 cells transfected with a plasmid encoding TRBP, pcDNA-TRBP (14), with and without silencing by siRNA-TRBP (Fig. ​(Fig.4G).4G). GFP silencing was not enhanced in HEp-2 cells overproducing TRBP, and it was not decreased by downregulating TRBP gene expression with siRNA-TRBP (data not shown). These results suggest that the high levels of TRBP in the cured cell lines are not the cause of the enhanced silencing in these cells.There was less TRBP protein in HEp-S31 (cl18) cells (2) than in HEp-2 and other control cells (IMR5) (Fig. ​(Fig.4H),4H), indicating that high levels of TRBP are not necessarily selected in cells persistently infected with PV. PACT was slightly downregulated in the cured cells (Fig. ​(Fig.4I).4I). Moreover, PACT is unlikely to be involved in the enhanced silencing in cured cells, because we used synthetic siRNAs and PACT functions principally during siRNA production by Dicer (14). We did not investigate the activities or subcellular distributions of the various factors involved in RNAi in the three cell lines, and they may differ. It is also possible that other factors, not tested here, contribute to the efficacy of siRNAs in cured cells. The molecular details of the mechanism involved remain to be determined.Overall, our results suggest that both a decrease in viral replication and the enhancement of gene silencing contributed to the mechanism by which cells persistently infected with poliovirus were cured by RNAi. Our results also indicate that cells displaying enhanced silencing may be selected during treatment with siRNAs. This may result in profound changes to cell phenotype, because RNAi plays an essential role in the regulation of cellular gene expression (15).     

病毒诱导的PVX cp转基因沉默及其DNA甲基化

利用PCR方法获得了马铃薯X病毒(PVX)外壳蛋白(CP)基因(cp),并将其构建到植物表达载体中,利用农杆菌介导的叶盘法转化烟草(Nicotiana tabacum L.)。Northern杂交及Run on实验表明有3株转基因烟草发生了转录后基因沉默。发生沉默的cp基因的甲基化分析结果表明,发生转录后基因沉默的cp基因发生了不同程度的甲基化,说明DNA甲基化并没有完全抑制cp基因的转录。利用PVX病毒对外壳蛋白正常表达的转基因烟草进行接毒,Northern杂交检测结果表明,病毒诱导cp发生了基因沉默。进一步的Run on结果表明,转基因烟草中cp基因在沉默前后转录速率并没有发生变化,说明病毒诱导的沉默是一种转录后沉默。对cp基因沉默前后的甲基化分析表明,病毒的侵染导致了cp基因甲基化程度的增加。     

Gene Transfer to Fish Cells by Attenuated Invasive Escherichia coli

Genetic immunization has proved effective in a number of applications including vaccination of rainbow trout (Oncorhynchus mykiss) against the fish pathogen infectious hematopoietic necrosis virus. However, injection vaccines, especially in aquaculture, are not as desirable as oral or immersion dosing schemes. In this report we present evidence that attenuated invasive Escherichia coli can infect and deliver plasmid DNA to salmonid fish cells.