Suppression of prion protein in livestock by RNA interference

Given the difficulty of applying gene knockout technology to species other than mice, we decided to explore the utility of RNA interference (RNAi) in silencing the expression of genes in livestock. Short hairpin RNAs (shRNAs) were designed and screened for their ability to suppress the expression of caprine and bovine prion protein (PrP). Lentiviral vectors were used to deliver a transgene expressing GFP and an shRNA targeting PrP into goat fibroblasts. These cells were then used for nuclear transplantation to produce a cloned goat fetus, which was surgically recovered at 81 days of gestation and compared with an age-matched control derived by natural mating. All tissues examined in the cloned fetus expressed GFP, and PCR analysis confirmed the presence of the transgene encoding the PrP shRNA. Most relevant, Western blot analysis performed on brain tissues comparing the transgenic fetus with control demonstrated a significant (>90%) decrease in PrP expression levels. To confirm that similar methodologies could be applied to the bovine, recombinant virus was injected into the perivitelline space of bovine ova. After in vitro fertilization and culture, 76% of the blastocysts exhibited GFP expression, indicative that they expressed shRNAs targeting PrP. Our results provide strong evidence that the approach described here will be useful in producing transgenic livestock conferring potential disease resistance and provide an effective strategy for suppressing gene expression in a variety of large-animal models.     

RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model

Huntington's disease (HD) is a fatal, dominant neurogenetic disorder. HD results from polyglutamine repeat expansion (CAG codon, Q) in exon 1 of HD, conferring a toxic gain of function on the protein huntingtin (htt). Currently, no preventative treatment exists for HD. RNA interference (RNAi) has emerged as a potential therapeutic tool for treating dominant diseases by directly reducing disease gene expression. Here, we show that RNAi directed against mutant human htt reduced htt mRNA and protein expression in cell culture and in HD mouse brain. Importantly, htt gene silencing improved behavioral and neuropathological abnormalities associated with HD. Our data provide support for the further development of RNAi for HD therapy.     

短发夹状双链RNA抑制丙型肝炎病毒IRES介导的基因表达

目的研究载体表达的短发夹状双链RNA（shRNA）对丙型肝炎病毒（HCV）IRES介导的基因表达的特异性抑制作用。方法构建HCV IRES调控的绿色荧光蛋白表达载体（pIRES—GFP）和虫荧光索酶表达载体（p5′ UTR—Luc），以及针对HCV IRES的shRNA表达载体（pshRNA-HCV）。共转染HepG2细胞，于转染后24、48、72h观察绿色荧光的强弱，用Western blot检测绿色荧光蛋白的表达，半定量逆转录聚合酶链反应法检测GFP的mRNA水平。双荧光索酶系统检测虫荧光索酶活性。结果pshRNA-HCV作用组绿色荧光强度明显弱于未干扰组，GFP蛋白表达量及虫荧光素酶活性降低60％～70％，半定量逆转录聚合酶链反应显示pshRNA-HCV导致了GFP基因mRNA水平的降低。结论针对HCV IRES的shRNA能够显著和特异地抑制该区域调控的蛋白表达水平及mRNA水平，该研究结果为利用RNA干扰技术治疗HCV感染进行了初步探索。     

Suppression of glucosylceramide synthase by RNA interference reverses multidrug resistance in human breast cancer cells

Glucosylceramide synthase (GCS), the enzyme that converts ceramide to glucosylceramide, induce multidrug resistance (MDR) in cancer cells. Recently, RNA interference (RNAi) is a powerful strategy for gene therapy by introducing double-stranded RNA and leading to the sequence-specific destruction. We have designed two different short hairpin RNAs (shRNAs) targeting GCS and introduced them into adriamycin- resistant human breast cancer cells (MCF-7/AdrR cells) to inhibit GCS expression. The results demonstrated that the shRNAs targeting GCS decreased GCS mRNA, abolished GCS protein levels and restored the sensitivity of MCF-7/AdrR cells to several antineoplastic drugs. This study revealed that this approach can reverse MDR effectively and it may be applicable to cancer patients as a specific means to restore the sensitivity to chemotherapy.     

转铁蛋白受体介导的Bcl-2shRNA转移对HL-60细胞生长的抑制作用

目的 探讨Bcl一2短发夹状RNA（shRNA）表达载体对白血病细胞HL-60生长的抑制作用。方法 采用转铁蛋白-多聚乙烯亚胺介导的转染方法将携带绿色荧光蛋白基因的重组Bcl-2 shRNA1、shRNA2表达载体转入HL-60细胞。通过荧光显微镜和流式细胞仪观察绿色荧光蛋白的表达；采用台盼蓝拒染法计数活细胞；用免疫细胞化学方法检测Bcl-2蛋白表达水平。结果 Bcl-2 shRNA1、shRNA2载体分别转入HL-60细胞后，细胞中Bcl-2蛋白表达水平均显著降低（P〈0．05），转染后48、72、96h细胞生长明显受到抑制。结论 转铁蛋白受体介导的Bcl-2 shRNA可显著抑制HL-60细胞生长。     

RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells

RNA interference (RNAi) is a biological process in which a double-stranded RNA directs the silencing of target genes in a sequence-specific manner. Exogenously delivered or endogenously encoded double-stranded RNAs can enter the RNAi pathway and guide the suppression of transgenes and cellular genes. This technique has emerged as a powerful tool for reverse genetic studies aimed toward the elucidation of gene function in numerous biological models. Two approaches, the use of small interfering RNAs and short hairpin RNAs (shRNAs), have been developed to permit the application of RNAi technology in mammalian cells. Here we describe the use of a shRNA-based live-cell microarray that allows simple, low-cost, high-throughput screening of phenotypes caused by the silencing of specific endogenous genes. This approach is a variation of "reverse transfection" in which mammalian cells are cultured on a microarray slide spotted with different shRNAs in a transfection carrier. Individual cell clusters become transfected with a defined shRNA that directs the inhibition of a particular gene of interest, potentially producing a specific phenotype. We have validated this approach by targeting genes involved in cytokinesis and proteasome-mediated proteolysis.     

Effect of vector-expressed shRNAs on hTERT expression

AIM: To study the effect of short hairpin RNAs (shRNAs) expressed from DNA vector on hTERT expression. METHODS: Oligonucleotides coding for four shRNAs against hTERT were cloned into a mammalian shRNA expression vector pUC18U6 to form pUC18U6ht1-4, which were then introduced into HepG2 cells by using liposome-mediated transfection. HepG2 cells transfected by pUC18U6 and pUC18U6GFPsir, which expressed shRNA against green fluorescent protein (GFP), were used as controls. hTERT mRNA in the transfected cells were quantified by using real-time fluorescent RT-PCR. RESULTS: Among the four shRNAs against hTERT, two decreased the hTERT mRNA level. Compared with the controls, pUC18U6ht which expressed the two shRNAs reduced hTERT mRNA by 39% and 49% (P<0.05). CONCLUSION: hTERT expression is inhibited by the shRNAs expressed from the DNA vector.     

Reversible suppression of an essential gene in adult mice using transgenic RNA interference

RNAi has revolutionized loss-of-function genetics by enabling sequence-specific suppression of virtually any gene. Furthermore, tetracycline response elements (TRE) can drive expression of short hairpin RNAs (shRNAs) for inducible and reversible target gene suppression. Here, we demonstrate the feasibility of transgenic inducible RNAi for suppression of essential genes. We set out to directly target cell proliferation by screening an RNAi library against DNA replication factors and identified multiple shRNAs against Replication Protein A, subunit 3 (RPA3). We generated transgenic mice with TRE-driven Rpa3 shRNAs whose expression enforced a reversible cell cycle arrest. In adult mice, the block in cell proliferation caused rapid atrophy of the intestinal epithelium which led to weight loss and lethality within 8-11 d of shRNA induction. Upon shRNA withdrawal, villus atrophy and weight loss were fully reversible. Thus, shRpa3 transgenic mice provide an interesting tool to study tissue maintenance and regeneration. Overall, we have established a robust system that serves the purpose of temperature-sensitive alleles in other model organisms, enabling inducible and reversible suppression of essential genes in a mammalian system.     

shRNA干扰Rab9 GTPase表达对麻疹病毒野生株体外增殖的抑制作用

目的构建Rab9 GTPase短发夹RNA（shRNA）表达载体,观察其对Rab9 GTPase基因表达和麻疹病毒野生株体外增殖的抑制作用。方法参照GenBank中Rab9 GTPase基因序列设计合成2对Rab9 GTPase基因特异性shRNA,定向克隆人表达载体,构建重组表达载体,酶切鉴定和序列分析证实后脂质体法转染U937细胞,然后感染麻疹病毒野生株,逆转录聚合酶链反应（RT-PCR）和免疫印迹技术（Western blot）检测转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平;标准蚀斑试验测定病毒滴度;流式细胞仪检测细胞凋亡率的变化;RT-PCR检测转染细胞内双链RNA依赖蛋白激酶（PKR）和2′-5′寡腺甙酸合成酶（OAS-1）的mRNA水平。结果酶切和序列分析证实,成功构建了靶向Rab9 GTPase基因的shRNA表达载体。2个shRNAs均可特异性抑制U937细胞内Rab9 GTPase mRNA和蛋白质的表达,最高抑制率分别为（90.5±0.2）％和（92.1±0.3）％;蚀斑试验结果表明,shRNAs可以有效抑制麻疹病毒野生株体外增殖,其抑制率可达到90％以上;流式细胞仪检测转染后细胞的凋亡率无明显变化;RT-PCR检测PKR和OAs-1的mRNA水平转染前后无明显变化。结论成功构建Rab9 GTPase特异性shRNA表达载体。shRNAs通过特异性抑制Rab9 GTPase基因表达抑制麻疹病毒野生株体外增殖。     

短发夹状RNA抑制SMYD3基因在HepG2细胞中的表达

目的构建针对人SET-和MYND-结构域含有蛋白3(SMYD3)基因的短发夹状RNA (shRNA)表达载体pGenesil-1-s,观察其对人肝癌细胞株HepG2细胞SMYD3基因表达的特异性抑制效应。方法设计合成针对SMYD3 mRNA编码序列267～288、302～323靶位点之间的核苷酸片断,并定向克隆到仅表达EGFP的空载体pGenesil-1的U6启动子下,构建重组质粒pGenesil-1-s1、pGenesil-1-s2；不针对任何序列的重组质粒pGenesil-1-hk作为对照。通过脂质体介导转染人肝癌细胞株HepG2,应用逆转录聚合酶链反应,western blot蛋白免疫印迹法分别从mRNA、蛋白水平检测阻抑效应。结果酶切鉴定和测序结果证实了pGenesil-1-s1、pGenesil-1-s2、pGenesil-1-hk的成功构建,转染HepG2细胞后,pGenesil-1- s1、pGenesil-1-s2组SMYD3 mRNA和蛋白表达均明显下调,与pGenesil-1-hk组及pGenesil-1对照组比较差异有统计学意义(P＜0.01)。结论构建的重组质粒pGenesil-1-s1、pGenesil-1-s2能特异性抑制SMYD3 在HepG2细胞中的表达。     

联合转染Survivin shRNA和CD44v3shRNA对结直肠癌SW480细胞增殖和侵袭能力的影响

目的:探讨联合转染Survivin短发夹RNA(short hairpin RNA,shRNA)和CD44v3 shRNA对结直肠癌SW480细胞增殖和侵袭能力的影响.为结直肠癌的基因治疗提供实验依据.方法:设计并构建能够稳定转录shRNA并干扰Survivin和CD44v3分子表达的质粒,将其单独及联合转染结直肠癌S...     

Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro

AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA) expression vector. METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method. RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control. Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively. CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).     

靶向PCSK9基因shRNA慢病毒载体的构建及其对PCSK9基因表达的沉默

目的设计以PCSK9基因为靶点的短发夹状RNA（shRNA）,构建重组慢病毒载体并鉴定此RNA干扰体系对PCSK9基因表达的影响。方法将3条人PCSK9基因的shRNA片段插入至慢病毒载体pLentilox3．7,与pCDNA3-hPCSK9．FLAG质粒用Lipofectamine2000共转染293细胞,Western blotting法鉴定出最有效的shRNA。此重组质粒与pCre—VSV-G、pLoxp．CMV—R8．91经293细胞包装后,产生的重组慢病毒感染293细胞,48h后转染pCDNA3．hPCSK9-FLAG质粒,Western blotting法检测人PCSK9基因表达的情况。结果经双酶切鉴定,构建了PCSK9shRNA慢病毒载体pLentilox—hPC—SK9,鉴定出hshRNA-2为最有效的shRNA。重组的慢病毒能明显的抑制人PCSK9的表达。结论慢病毒介导的shRNA干扰技术可特异性的阻断PCSK9的表达,为进一步探讨PCSK9特异性的shRNA治疗脂代谢异常疾病奠定了基础。     

小鼠FOXP3-siRNA慢病毒载体的构建及其对小鼠动脉粥样硬化的影响

目的:构建小鼠FOXP3基因特异性siRNA慢病毒载体,并对其进行功能性研究.方法:根据GeneBank提供的小鼠FOXP3cDNA序列,设计4条RNA干扰靶点序列,制备双链DNA oligo,与制备好的双酶切慢病毒载体连接,再转入细菌感受态细胞DH-5a,行PCR鉴定出阳性克隆并测序,制备成FOXP3-siRNA慢病毒载体,利用Western-blot方法对构建的载体进行功能性研究;将载体通过尾静脉注入小鼠体内,观察其对小鼠动脉粥样硬化形成的影响.结果:构建的小鼠FOXP3基因siRNA慢病毒载体,经PCR和DNA测序证实与设计完全一致,并对Foxp3+CD4+CD25+调节性T细胞有显著敲减效应;其能显著促进动脉粥样硬化形成.结论:体内外实验表明,成功构建了小鼠FOXP3基因的特异性siRNA慢病毒载体.     

靶向XIAP的shRNA重组质粒的构建

目的:设计以X染色体连锁凋亡抑制蛋白(XIAP)为靶向的shRNA,构建携带此shRNA的重组质粒,检测其抑制XIAP表达的效应,筛选RNA干扰作用最强的shRNA片段.方法:设计4对针对XIAP基因不同位点的shRNA片段,构建携带此shRNA片段的真核表达载体psiRNA-Hhneo-XIAP,通过脂质体介导的方法将重组质粒转染到肝癌细胞株HepG2细胞中.采用逆转录酶链式反应(RT-PCR)及蛋白印迹(Western blotting)方法检测XIAP的mRNA及蛋白表达情况,比较转染前后其表达差异,以判断各shRNA的干扰效应.结果:成功构建含shRNA片段的重组质粒.经质粒测序证实,插入的DNA片段的序列与设计序列完全一致.重组质粒转染HepG2细胞后,XIAP基因的mRNA水平及蛋白表达水平明显下调,其中以1号重组质粒效应最强.shRNA作用48h后,对HepG2细胞中XIAP mRNA和蛋白的抑制率与3,4号重组质粒相比,均具有显著性差异(mRNA:94.5% vs 81.5%,82.6%,均P<0.01:蛋白:92.6% vs 80.7%,82.9%,均P<0.01).结论:成功构建了携带以XIAP为靶向的shRNA的重组质粒,其对肝癌细胞内XIAP的表达具有显著抑制效应.X染色体连锁凋亡抑制蛋白;;短发夹状RNA;;肝癌;;逆转录聚合酶链式反应;;免疫蛋白印迹