摘要
疯牛病是牛海绵状脑病(Bovine spongiform Eneephalopathy,BSE)的俗称,是由朊病毒引起的一种传染性的,慢性的,致死性的中枢神经系统退行性疾病。疯牛病是由Prnp基因编码的朊病毒蛋白PrPc发生构象变化为PrPsc而引起的传染性疾病, PrPsc在中枢神经的聚集引发了动物的传染性海绵状脑病。因此,降低(下调)PrPc的表达量使其减少向PrPsc转化,是抑制疯牛病的有效途径。RNAi是一种高效的特异性强的基因阻断技术,它可以特异性的降解细胞内与其序列同源的mRNA,封闭内源性基因表达,实现类似基因敲除的效果。因此本实验采取了RNA干涉的方法对疯牛病进行了基础研究。
siRNA表达载体法能够在产生细胞内持续产生siRNA,介导较长时间的基因沉默,且带有抗生素标记的基因,能够进行药物筛选,可以获得稳定转染的细胞,适合长期的实验研究。本文利用RNAi技术,针对牛源cDNA序列设计了3段PrPc干扰序列及一个阴性对照,经反转处理并加酶切位点和loop环等相关处理后连到空载体pGenesil-1上,构建了针对prnp的干扰载体,分别命名为Prnp1,Prnp2,Prnp3和Prnp-NC。然后将4个干扰载体分别转染牛胎儿成纤维细胞,转染后的第2天,第3天分别收集瞬时转染的细胞,利用无菌通道的流式细胞仪分选出带荧光的细胞。用实时荧光定量PCR方法和进行Western blot法对其进行干扰效率的检测。结果表明,3个干扰片段的抑制效率分别是51.2%、85.7%、74.6%,阴性对照的干扰效率是2.05%,因而得到了一个较为有效的干扰片段prnp2。然后用700μg/mL G418对转染prnp2的牛胎儿成纤维细胞进行药物筛选,15 d之后获得了稳定转染的细胞单克隆。
本实验结合无菌通道的流式细胞仪技术,在分选出瞬时转染的阳性细胞后进行干扰效率的检测,避免了细胞转染效率低对干扰效率检测的影响。在筛选出有效的干扰片段后进行了稳定转染,筛选出的细胞可以用于后续长期的实验研究。
The mad cow disease is the Bovine spongiform Eneephalopathy’s(BSE) popular name. It is an infectious, chronic, lethal central nervous system recession disease which is caused by the prion protein– PrPsc. PrPsc is the isoform of normal prion protein PrPc, they are coded by prnp gene. Along with the aggregation of PrPsc in the central nervous system, it can result to the transmissible bovine spongiform encephalopathy. By cutting down PrPc’s expression to reach the purpose of decreasing its conversion into PrPsc is an effective way to inhibit BSE. RNAi is a highly efficient and specific gene knockdown technology, it can specially degrad it’s homology sequence’s mRNA in the cells, inhibit endogenous gene expression, then achieve the effect similar to gene knockout. So we employed RNA interference technology to knockdown prnp expression, it will contribute to deep research of this gene.
SiRNA expression vector can continuously generate siRNA in the cells, make a long time gene silencing. And because it carries the antibiotic resistance gene, we can conduct drug screening to get the stably transfected cells, this is suitable for long time research. In this article, we used the technology of RNAi to cut down PrPc’s expression, three candidate shRNAs targeting the CDS of bovine prnp gene and a negative control were designed, after reverse transcription, restriction enzyme cutting site and loop structure introduction, they were ligated into the vector of pGenesil-1 respectively, then we successfully constructed 4 RNAi-vector targeted to prnp, they were termed Prnp1, Prnp2, Prnp3 and Prnp–NC respectively. We transfected the four vectors into bovine fibroblast cells separately, 2 days and 3 days after transfection, we collected the transient transfected cells with fluorescence by flow cytometry(FCM)in sterile channel, then examined their suppression efficiency by the means of Real-time PCR and Western blot, the results showed that the interference effects of the three shRNA vectors were 51.2%、85.7% and 74.6%, the negative control’s efficiency is 2.05%, so we obtained a more efficacious shRNA―Prnp2. After that, we used 700μg/mL G418 selected the fibroblast cells transfected with Prnp2, after 15 days, the positive cell clones were obatained.
In this study, we combined the technology of FCM selected the transient transfected positive cells, then detected their interference efficiency, this can avoid the side effect of lower transfection efficiency. Except that, we obtained positive cell clones by the means of G418 selection, it provides a way for later study.
引文
宝明涛.2008.利用RNAi抑制小鼠胎儿成纤维细胞中FGFS基因的表达. [硕士学位论文].内蒙古:内蒙古大学
樊鹏程,贾正平,马骏,王荣. 2008. siRNA设计研究进展及在线设计.细胞与分子免疫学杂志, 24(2): 202~203
管峰,潘磊,石国庆,刘守仁,杨利国. 2009.绵羊PRNP遗传多样性与抗病育种研究进展.遗传,31(2):137~141
阚学峰,高英堂. 2004. siRNA四种制备技术的介绍.国外医学临床生物化学与检验学分册, 25(4): 342~344
廖延熊. 1999.朊粒病.畜牧与兽医,31(3):1~4
刘默芳,蒋帅,王恩多. 2007. RNAi机器.生物化学与生物物理进展,34(10): 1012~1017
史毅,金由辛. 2008. RNA干扰与siRNA(小干扰RNA)研究进展.生命科学,20(2):196~201
王海嵘,顾春红,钟济华,王婷,韩洁英,陈芳源,欧阳仁荣. 2006.流式细胞术结合G418筛选重组质粒稳定转染细胞.诊断学理论与实践, 5(1): 52~55
王少华,李宁. 2006.朊蛋白基因敲除无启动子打靶载体的构建策略.广西农业生物科学, 25: 91~96
杨蕴天,江新梅,胡轶虹,林世和. 2008.朊蛋白与分子伴侣.中国人兽共患病学报,24(1):87~97
尹秀山,张令强,贺福初. 2006. RNAi技术在转基因动物中的应用.遗传, 28(3): 351~356
Agrawal N, Dasaradhi P V N, Mohmmed A, Malhotra P, Bhatnagar R K, Mukherjee S K. 2003. RNA Interference: Biology, Mechanism, and Applications. Microbiology and Molecular Biology Reviews, 67(4): 657~685
Aguzzi A, Heikenwalder M, Miele G. 2004. Progress and problems in the biology, diagnostics and therapeutics of prion diseases. J Clin Invest, 114(2): 153~160
Aguzzi A, Sigurdson C, Heikenwaelder M. 2008. Molecular Mechanisms of Prion Pathogenesis. Annu Rev Pathol Mech Dis, 3: 11~40
Alper T, Cramp W A, Haig D A, Clarke M C. 1967. Does the agent of scrapie replicate without nucleic acid. Nature, 214: 764~766
Amarzguioui M, Prydz H. 2004. An algorithm for selection of functional siRNA sequences. Biochem Biophys Res Commun, 316: 1050~1058
Ballerini C, Gourdain P, Bachy V, Blanchard N, Levavasseur E, Gregoire S, Fontes P, Aucouturier P, Hivroz C, Carnaud C. 2006. Functional Implication of Cellular Prion Protein in Antigen-Driven Interactions between T Cells and Dendritic Cells. J Immunol, 176(12): 7254~7262
Bantounas I, Phylactou1 L A, Uney J B. 2004. RNA interference and the use of small interfering RNA to study gene function in mammalian systems. Journal of Molecular Endocrinology, 33: 545~557
Barbot C, Castro L, Oliveira C, Carpenter S. 2010. Varient Creutzfeldt-Jakob disease: the first confirmed case from Portugal shows early onset, long duration and unusual pathology. J Neurol Neurosurg Psychiatry, 81(1): 112~114
Bosques C J, Imperiali B. 2003. The interplay of glycosylation and disulfide formation influencesfibrillization in a prion protein fragment. Proc Natl Acad Sci USA, 100(13): 7593~7598
Bounhar Y, Mann K K, Roucou X, LeBlanc A C. 2006. Prion protein prevents Bax-mediated cell death in the absence of other Bcl-2 family members in Saccharomyces cerevisiae. FEMS Yeast Res, 6(8): 1204~1212
Brandner S, Isenmann S, Raeber A J, Fischer M B, Sailer A, Kobayashi Y, Marino S, Weissmann C,Aguzzi A. 1996. Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature, 379: 339~343
Brodsky I E,Medzhitov R. 2009. Targeting of immune signaling networks by bacterial pathogens.Nat Cell Biol, ll: 521~526
Bruce M E, Will R G, Ironside J W. 1997. Transmissions to mice indicate that‘new variant’CJD is caused by the BSE agent. Nature, 389: 498~501
Brummelkamp T R, Bernards R, Agami R. 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science, 296: 550~553
Bueler H, Aquzzi A, Sailer A, Greiner R A, Autenried P, Aquet M,Weissmann C. 1993, Mice devoid of PrP are resistant to scrapie. Cell, 73: 1339~1347
Büeler H, Fischer M, Lang Y, Bluethmann H, Lipp H P , DeArmond S J, Prusiner S B, Aguet M, Weissmann C. 1992. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature, 356: 577~582
Caughey B. 2003. Prion protein conversions: insight into mechanisms, TSE transmission barriers and strains. British Medical Bulletin, 66: 109~120
Cioca D P, Aoki Y, Kiyosawa K. 2003. RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines. Cancer Gene Ther, 10(2): 125~133
Clemens J C, Worby C A, Simonson-Leff N, Muda M, Maehama T, Hemmings B A, Dixon J E. 2000. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Nati Acad Sci, 97(12):6499~6503
Deleault N R, Lucassen R W, Supattapone S. 2003. RNA molecules stimulate prion protein conversion. Nature, 425: 717~720
Deleault N R, Lucassen R W, Supattapone S. 2003. RNA molecules stimulate prion protein conversion. Nature, 425: 717~720
Denti M A, Rosa A, Sthandier O, Angelis F G, Bozzoni I. 2004. A new vector, based on the PolⅡpromoter of the U1 snRNA gene, for the expression of siRNAs in mammalian cells. Mol Ther, 10: 191~199
Gains M J, Roth K A, LeBlanc A C. 2006. Prion protein protects against ethanol-induced Bax-mediated cell death in vivo. Neuroreport, 17(9): 903~906
Gajdusek D C, Gibbs C J Jr, Asher D M, David E. 1968. Transmission of experimental Kuru to the spider monkey (Ateles geoffreyi). Science, 162 (854) : 699~694
Gallozzi M, Chapuis J, Provost F L, Dur A L, Morqenthaler C, Peyre C, Daniel Carlier N, Pailhoux E , Vilotte M, Passet B, Herzog L, Beringue V, Costa J, Tixador P, Tilly G, Laude H , Vilotte J L. 2008. Prnp knockdown in transgenic mice using RNA interference. Transgenic Resarch, 17(5): 783~791
Glover J R, Kowal A S, Schirmer E C, Patino M M, Liu J J, Lindquist S. 1997. Self-seeded fibers formed by Sup35,the protein determinant of[PSI+],a heritable prion-like factor of S.cerevisiae. Cell, 89: 811~819
Goldfarb L G, Cervenakova L, Gajdusek D C. 2004. Genetic studies in relation to kuru: an overview. Curr Mol Med, 4(4):375~384
Golding M C, Long C R, Carmell M A, Hannon G J, Westhusin M E. 2006. Suppression of prion protein in livestock by RNA interference. Proc Natl Acad Sci USA, 103: 5285~5290
Gregory R, Chendrimada T, Cooch N, Shiekhattar R. 2005. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell, 123(4): 631~640
Guo Y, Zhang C S, Li S G, Li F, Gu R H, Xing F Y, Li Y, Yao G, Chen X J. 2009. RNAi-mediated Stable Silencing of HGPRT Expression in Rabbit Fibroblasts and SCNT Embryo Production. Progress in Biochemistry and Biophysics, 36(7): 872~879
Hachiya N S, Sakasegawa Y, Sasaki H, Jozuka A, Tsukita S, Kaneko K. 2004. Oligomeric Aip2p/ Dld2p modifies the protein conformation of bot h properly folded and misfolded subst rates in vitro. Biochem Biophys Res Commun, 323: 339~344
Hannon G J. 2002. RNA interference. Nature, 418(11):244~251
Harper J D, Lansbury P T Jr. 1997. Models of amyloid seeding in Alzheimerps disease and scrapie : mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu Rev Biochem, 66: 385~407
Heppner F L, Aguzzi A. 2004. Recent development s in prion immuno-therapy. Curr Opin Immunol, 16(5): 594~598
Hijazi N, Shaked Y, Rosenmann H, Ben-Hur T, Gabizon R. 2003. Copper binding to PrPc may inhabit prion disease propagation. Brain Res, 993(1-2):192~200
Hills D, Comincini S, Schlaepfer J, Dolf G, Ferretti L, Williams J L. 2001. Complete genomic sequence of the bovine prion gene (PRNP)and polymorphism in its promoter region. Anim Genet, 32(4): 231~232
Hu W, Kieseier B, Frohman E, Eagar T N, Rosenberg R N, Hartung H P, Stüve O. 2008. Prion proteins: physiological functions and role in neurological disorders. J Neurol Sci, 264(1-2):1~8
Ivanova L, Barmada S, Kummer T, Harris D A. 2001. Mutant prion proteins are partially retained in the endoplasmic reticulum. J Biol Chem, 276: 42409~42421
Jarrett J T, Lansbury P J. 1993. Seeding“one-dimensional crystallization”of amyloid: a pathogenic mechanism in Alzheimer’s disease and scrapie? Cell, 73: 1055~1058
Karpuj M V, Giles K, Gelibter-Niv S, Scott M R, Lingappa V R, Szoka F C, Peretz D, Denetclaw W, Prusiner S B. 2007. Phosphorothioate oligonucleotides reduce PrP levels and prion infectivity in cultured cells. Mol Med, 13: 190~198
Kim D H, Longo M, Han Y, Lundberg P, Cantin E, Rossi J J. 2004. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nature Biotechnology, 22: 321~325
Kim Y, Han B, Titlowa W, Maysa C E, Kwonb M, Ryoua C. 2009. Utility of RNAi-mediated prnp gene silencing in neuroblastoma cells permanently infected by prions: Potentials and limitations. Antiviral Research, 84 : 185~193
Lambeth L S, Moore1 R J, Muralitharan M, Dalrymple B P, McWilliam S, Doran J T. 2005. Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs. BMC Biotechnology, 5: 13~22
Laurent M. 1996. Prion disease and the“protein only”hypothesis: a theoretical dynamic study. Biochem J, 318: 35~39
Lee C, Yu M H. 2005. Protein Folding and Diseases. Journal of Biochemistry and Molecular Biology, 38(3): 275~280
Lee I Y, Westaway D, Smit A F, Wang K, Seto J, Chen L, Acharya C, Ankener M, Baskin D, Cooper C, Yao H, Prusiner S B, Hood L E. 1998. Complete genomic sequence and analysis of the prion protein gene region from three mammalian species. Genome Res, 8(10): 1022~1037
Lee Y, Hur I, Park S Y, Kim Y K, Suh M R , Kim V N. 2006. The role of PACT in the RNA silencing pathway. EMBO J, 25: 522~532
Leuschner P, Ameres S, Kueng S, Martinez J. 2006. Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep, 7(3): 314~320
Lima L M, Cordeiro Y, Tinoco L W, Marques A F, Oliveira C L, Sampath S, Kodali R, Choi G, Foguel D, Torriani I, Caughey B, Silva JL. 2006. Structural insights into the interaction between prion protein and nucleic acid. Biochemistry, 45(30): 9180~9187
Lingel A, Simon B, Izaurralde E, Sattler M. 2004. Nucleic acid 3′-end recognition by the Argonaute2 PAZ domain. Nat Struct Mol Biol, 11(6): 576~577
Lipardi C, Wei Q, Paterson B M. 2001. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell, 107(3): 297~307
Liu M, Yu S, Yang J, Yin X, Zhao D. 2007. RNA and CuCl2 induced conformational changes of the recombinant ovine prion protein. Molecular and Cellular Biochemistry, 294(1-2):197~203
Liu Q H, Rand T A, Kalidas S, Du F H, Kim H E, Smith D P, Wang X D. 2003. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science, 301(5641): 1921~1925
L?tscher M, Recher M, Lang K S, Navarini A, Hunziker L, Santimaria R, Glatzel M, Schwarz P, B?ni J, Zinkernagel R M. 2007. Induced prion protein controls immune-activated retroviruses in the mouse spleen. PLoS ONE, 2(11): 1185
Ma J B, Ye K, Patel D J. 2004. Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature, 429(6989): 318~322
Mabbott N A, Macpherson G. 2006. Prions and their lethal journey to the brain. Nature Rev Microbiol, 4(11): 201~211
Makarava N, Kovacs G G, Bocharova O, Savtchenko R, Alexeeva I, Budka H, Robert G, Baskakov I V. 2010, Recombinant prion protein induces a new transmissible prion disease in wild-type animals. Acta neuropathologica,119(2):177~187
Málaga-Trillo E, Solis G P, Schrock Y, Geiss C, Luncz L, Thomanetz V, Stuermer C A O. 2009. Regulation of embryonic cell adhesion by the prion protein.PLos Biol, 7: 576~590
Mallucci G, Dickinson A, Linehan J, Klohn P C, Brandner S, Collinge J. 2003. Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. Science, 302: 871~874
Maranga C, Tomari Y, Shin C, Bartel D, Zamore P. 2005. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell, 123(4): 607~620
Mello C C. 2007. Return to the RNAi World: Rethinking Gene Expression and Evolution. Angew Chem Int Ed, 46: 6985~6994
Miyagishi M, Taira K. 2002. U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnology, 20: 497~500
Moore R C, Lee I Y, Silverman G L, Harrison P M, Strome R, Heinrich C, Karunaratne A, Pasternak S H,
Chishti M A, Liang Y, Mastrangelo P, Wang K, Smit A F, Katamine S, Carlson G A, Cohen F E, Prusiner S B, Melton D W, Tremblay P, Hood L E, Westaway D. 1999. Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein Doppel. J Mol Biol, 292(4): 797~817
Mummery-Widmer J L, Yamazaki M, Stoeger T, Novatchkova M, Bhalerao S, Chen D, Dietzl G, Dickson BJ, Knoblich J A. 2009. Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi. Nature, 458(7241):987~992
Nagy P, Arndt-Jovin D J, Jovin T M. 2003. Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbB1 -overexpressing cells. Exp Cell Res, 285(1): 39~49
Nykanen A, Haley B, Zamore P D. 2001. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell, 107(3): 309~321
Oliveira S, Storm G, Schiffelers R M. 2006. Targeted Delivery of siRNA. Journal of Biomedicine and Biotechnology, 2006: 1~9
Paitel E, Fahraeus R, Checler F. 2003. Cellular prion protein sensitizes neurons to apoptotic stimuli through Mdm2-regulated and p53-dependent caspase 3-like activation. J Biol Chem, 278: 10061~10066
Pantera B, Bini C, Cirri P, Paoli P, Camici G, Manao G, Caselli A. 2009. PrPc activation induces neurite outgrowth and differentiation in PC12 cells: role for caveolin-1 in the signal transduction pathway. J Neurochem, 110(1):194~207
Pezza J A, Langseth S X, Raupp Yamamoto R, Doris S M, Ulin S P, Salomon A R, Serio T R. 2009. The NatA acetyltransferase couples Sup35 prion complexes to the [PSI+] phenotype. Mol Biol Cell, 20(3): 1068~1080
Prusiner S B, Scott M , Foster D, Pan K M, Groth D, Mirenda C, Torchia M, Yang S L, Serban D, Carlson C A, Hoppe P C, Westaway D, DeArmond S J. 1990. Transgenetic studies implicate interaction between homologous PrP isoformin scraple prion replication. Cell, 63(4):673~686.
Prusiner S B. 1982. Novel proteinaceous infectious particles cause scrapie. Science, 216: 136~144
Prusiner S B. 1997. Prion diseases and the BSE crisis. Science, 278(10): 245~251
Prusiner S B. 1998. Prions. Proc Natl Acad Sci USA, 95: 13363~13383
Richt J A, Kasinathan P, Hamir A N, Castilla J, Sathiyaseelan T, Vargas F, Sathiyaseelan J, Wu H, Matsushita H, Koster J, Kato S, Ishida I, Soto C, Robl J M , Kuroiwa Y. 2007. Production of cattle lacking prion protein. Nature Biotechnology, 25(1):132~138
Rivas F V, Tolia N H, Song J J, Aragon J P, Liu J, Hannon G J, Joshua-Tor L. 2005. Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol, 12(4): 340~349
Rossi D, Cozzio A, Flechsig E, Klein M A, Rülicke T, Aguzzi A, Weissmann C. 2001. Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain. EMBO J, 20(4): 694~702
Rossi L, Arciello M, Capo C, Rotilio G. 2006. Copper imbalance and oxidative stress in neurodegeneration. Ital J Biochem, 55(3-4): 212~221
Roucou X, Gains M, LeBlanc A C. 2004. Neuroprotective Functions of Prion Protein. J Neurosci Res, 75(2): 153~161
Roucou X, Giannopoulos P N, Zhang Y, Jodoin J, Goodyer C G, LeBlanc A. 2005. Cellular prion proteininhibits proapoptotic Bax conformational change in human neurons and in breast carcinoma MCF-7 cells. Cell Death and Differentiation, 12: 783~795
Sakaguchi S. 2009. Prospects for preventative vaccines against prion diseases Protein. Pept Lett, 16(3): 260~270
Sassoon J, Sadowski M, Wisniewski T, Brown D R. 2005. Therapeutics and prion disease: can immunisation or drugs be effective? Mini Rev Med Chem, 5(4): 361~366
Schatzl H M, Costa M D, Taylor L, Cohen F E, Prusiner S B. 1995. Prion protein gene variation among primates. J Mol Biol, 245(4): 362~374
Silva J L, Lima L M, Foguel D, Cordeiro Y. 2008. Intriguing nucleic-acid-binding features of mammalian prion protein. Trends in Biochemical Sciences, 33(3):132~140
Singh A, Kong Q, Luo X, Petersen R B, Meyerson H, Meyerson H, Singh N. 2009. Prion Protein (PrP) Knock-Out Mice Show Altered Iron Metabolism: A Functional Role for PrP in Iron Uptake and Transport. PLoS ONE, 4(7): 6115
Soares-Fernandes J P, Ribeiro M, Machado A. 2009. Hot Cross Bun" Sign in Variant Creutzfeldt-Jakob Disease. Am J Neuroradiol, 30: 37
Stuermer C A O, Langhorst M F, Wiechers M F, Legler D F, Hanwehr S H, Guse A H, Plattner H. 2004. PrPc capping in T cells promotes its association with the lipid raft proteins reggie-1 and reggie-2 and leads to signal transduction.FASEB J, 18: 1731~1733
Sui G C, Soohoo C, Affar E B, Gay F, Shi Y J, Forrester W C, Shi Y. 2002. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. PNAS, 99(8): 5515~5520
Supattapone S. 2004. Prion protein conversion in vitro. Journal of molecular medicine, 82(6): 348~356
Sutou S, Kunishi M, Kudo T, Wongsrikeao P, Miyagishi M, Otoi T. 2007. Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology. BMC Biotechnology, 7: 44
Tanaka M, Chien P, Yonekura K, Weissman J S. 2005. Mechanism of cross-species prion transmission: an infectious conformation compatible with two highly divergent yeast prion proteins. Cell, 121: 49~62
Tillmann H L, Zender S, Mundt B, Waltemathe M, G?sling T, Flemming P, Malek N P, Trautwein C, Manns M P, Kühnel F, Kubicka S. 2003. Caspase 8 small interfering RNA p revents acute liver failure in mice. Proc Natl Acad Sci USA, 100(13): 7797~7802
Tolia N H, Joshua-Tor L. 2007. Slicer and the Argonautes. Nature Chemical Biology, 3(1): 36~43 Trevitt C R, Collinge J. 2006. A systematic review of prion therapeutics in experimental models. Brain, 129: 2241~2265
Trushina E, Du Charme J, Parisi J, McMurray C T. 2006. Neurological abnormalities in caveolin-1 knocked out mice.Behav Brain Res, 172(1): 24~32
Uppington K M, Brown D R. 2008. Resistance of cell lines to prion toxicity aided by phospho-ERK expression.J Neurochem, 105(3): 842~852
Verca M S B, Weber P, Mayer C, Graf C, Refojo D, Kühn R, Grummt I, Lutz B. 2007. Development of a species-specific RNA polymerase I-based shRNA expression vector. Nucleic Acids Res, 35(2): 10
Vorberg I, Priola S A. 2002. Molecular basis of scrapie strain glycoform variation,J Biol Chem, 277: 36775~36781
Watt N T, Routledge M N, Wild C P, Hooper N M. 2007. Cellular prion protein protects against reactive-oxygen-species-induced DNA damage. Free Radic Biol Med, 43: 959~967
Weissmann C, Enari M, Klohn P C, Rossi D, Flechsig E. 2002. Transmission of prions. Proc Natl Acad Sci USA, 99: 16378~16383
Weissmann C, Aguzzi A. 2005. Approaches to therapy of prion diseases. Annu Rev Med, 56: 321~344
Westerhout E M, Berkhout B. 2007. A systematic analysis of the effect of target RNA structure on RNA interference. Nucleic Acids Res, 35: 4322~4330
Wickner R B. 1994. [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science, 264: 566~569
Wong C, Xiong L W, Horiuchi M, Raymond L, Wehrly K, Chesebro B, Caughey B. 2001. Sulfated glycans and elevated temperature stimulate PrP(sc)-dependent cell-free formation of protease-resistant prion protein. EMBO J, 20(3): 377~386
Zhang L, Yang N, Mohamed-Hadley A, Rubin S C, Coukos G. 2003. Vector-based RNAi, a novel tool for isoform specific knock-down of EGF and antiangiogenesis gene therapy of cancer. Biochem Biophys Res Commun, 303(4): 1169~1178
Zomosa-Signoret V, Arnaud J D, Fontes P, Alvarez-Martinez M T, Liautard J P. 2008. Physiological role of the cellular prion protein. Vet Res, 39(4):9