Dppa2基因在维持小鼠胚胎干细胞不分化过程中的功能研究及其启动子的克隆与转录调控分析
|
摘要
Dppa2 (Developmental pluripotency-associated 2)是近年发现的在多能性细胞中特异表达的新基因。目前关于DPPA2(人)Dppa2(小鼠)基因的相关信息主要包括两方面：DPPA2/Dppa2作为一个多能性标志基因的表达分析和DPPA2作为一个cancer/testis基因在部分癌细胞中的表达。然而DPPA2/Dppa2基因的功能和发挥作用的机制仍有待进一步的研究。本课题的目的就在于以小鼠胚胎干细胞(mouse embryonic stem cells, mESCs)为材料,探索Dppa2基因在维持mESCs不分化过程中所发挥的作用以及可能的分子机制。
我们首先利用Real-time PCR分析了Dppa2基因在mESCs分化过程中的表达水平变化情况,并将之与Oct4, Sox2和Nanog基因的表达变化情况进行了分析和比较。我们采用了两种使mESCs分化的方法：悬浮培养形成拟胚体(Embryoid bodies, EBs)和维甲酸(A11trans-retinoic acid, atRA)诱导mESCs分化。我们的结果表明,在两种分化条件下,Dppa2基因均表现出和Oct4, Sox2或Nanog基因相似的表达变化趋势,在分化早期时间点均出现不同程度的表达上调,而在mESCs分化后期各基因表达水平均显著下调至非常低的水平。
为了获得理想的mESCs转染效率,实现载体法对Dppa2基因的RNA干扰(RNA interference, RNAi),我们对脂质体LipofectamineTM2000转染mESCs的条件进行了摸索和优化。结果提示低密度培养条件下(30-50%)的mESCs更容易获得较高的转染效率(≥50%)。
利用RNAi技术我们首次对mESCs中Dppa2基因进行了表达抑制。RT-PCR和Real-time PCR结果表明我们的RNAi是成功的,两个针对Dppa2基因的干扰组中干扰效率分别达到了85%(sh1)和78%(sh2)。Dppa2基因的表达被抑制后mESCs表现出了一定的分化趋势,表现为：碱性磷酸酶(Alkaline phosphatase, AKP)活性下降,染色有所减弱；多能性标记基因Oct4和Nanog的表达水平出现了微弱的下调(下调约20%左右)以及部分胚层标志基因(如内胚层的Fst和滋养外胚层的Psx1基因)表达出现不同水平的上调。此外,通过BrdU嵌入实验对细胞的增殖能力变化进行检测,结果表明,Dppa2基因的表达抑制引起了mESCs增殖能力明显下降。端粒酶活性检测结果显示Dppa2基因的表达抑制并未引起mESCs端粒酶活性的明显改变。
利用5'-RACE技术,我们将Dppa2基因的转录起始位点(Transcriptional start site, TSS)确定在其ATG上游-30bp处。此外,利用双荧光素酶报告系统,我们首次分析和检测了Dppa2基因不同启动子候选区段的活性,结果提示-176bp-+293bp区域为Dppa2核心启动子所在区。Dppa2启动子在mESCs中具有高启动活性,在P19细胞(小鼠胚胎癌细胞系,mECCs)中有一定启动活性,而在我们目前所检测的其它几种细胞中均基本无启动活性,提示Dppa2启动子在多能性细胞中特异表达。
通过生物信息学分析我们发现在Dppa2基因上游存在多个Oct4转录因子保守识别位点(ATGC(A/T)(A/T)-(A/T)),提示该基因可能受到Oct4的转录调控。进一步通过染色质免疫共沉淀(chromatinimmunoprecipitation, ChIP)和凝胶电泳迁移率变动分析(electrophoretic gel mobility shift assay, EMSA)等实验验证,我们首次确定了Dppa2基因上游-1964--1950处为Oct4转录调控识别位点。
总之,我们的研究结果表明Dppa2基因在维持mESCs不分化过程中发挥着一定的作用。此外,我们首次通过实验分析了该基因的转录起始位点并分离得到其启动子,并证实该基因受到了Oct4的(正)转录调控,提示Dppa2基因可能通过作为Oct4的下游基因之一参与维持mESCs不分化状态。我们的结果为进一步阐明维持胚胎干细胞不分化状态的分子调控机制奠定良好的基础。
Dppa2 (Developmental pluripotency-associated 2) is a recently identified gene specifically expressed in pluripotent cells. Current information on DPPA2 (human)/Dppa2 (mouse) were collected from the expression analysis of DPPA2/Dppa2 as a pluripotent marker and DPPA2/Dppa2 expression in cancer cells as a cancer/testis gene. Despite the current information, a further study in DPPA2/Dppa2 function as well as the mechanisms is needed. Our study is aimed at exploring the role of Dppa2 in maintaining the mESCs undifferentiation and the possible mechanisms.
Firstly, we used Real-time PCR to analyze the change of Dppa2 expression during mESCs differentiation, and compared the change with those of Oct4, Sox2 and Nanog. We adapted two methods to induce mESCs differentiation:suspension culture to form embryoid bodies (EBs) and all trans-retinoic acid (atRA) induction. Our results showed that, under the two differentiation conditions, Dppa2 displayed a similar trend of expression change to Oct4, Sox2 and Nanog:The expression was upregulated in earlier time then significantly downregulated to a very low level in later period during mESCs differentiation.
To achieve ideal transfection efficiency on mESCs and a successful RNA interference (RNAi) on Dppa2, we investigated and optimized the conditions of mESCs transfection with LipofectamineTM2000. Our results suggest that a low confluence of mESCs (30-50%) is prone to result in a relatively high transfection efficiency (≥50%).
For the first time, we used RNAi to inhibit Dppa2 expression in mESCs. The results of RT-PCR and Real-time PCR demonstrated that the RNAi on Dppa2 is successful, where the interference efficiencies in two inference groups shl and sh2 respectively reached 85% and 78%. After Dppa2 RNAi, mESCs showed considerable trends of differentiation, which indicated as a decreased AKP activity, a weak stain; slight downregulation (-20%) of Oct4 and Nanog expression as well as differently levels of upregulation on parts of germ layer marker genes (such as Fst of endoderm and Psx1 of Trophoblastic ectoderm). In addition, we detected the change of mESCs proliferation capability using BrdU assay. The results indicated that the inhibition of Dppa2 expression caused a significant decline on mESCs'proliferation capability.
We located the transcriptional start site (TSS) of Dppa2 at-30bp upstream its start codon ATG using 5'-RACE method. In addition, we used a dual luciferase reporter system to analyze and detect the activities of different candidate regions of Dppa2 promoter. The results suggested that-176bp-+293bp region is the basal promoter region of Dppa2. Dppa2 promoter has a high activity in mESCs and a certain activity in P19 (mouse embryo carcinoma cells, mECCs) while nearly no activity in other cell lines we have detected, suggesting the promoter has a specific activity in pluripotent cells.
We performed bioinformatics analysis and found that multiple recognition sites (ATGC(A/T)(A/T)-(A/T)) of Oct4 transcription factor exist in upstream region of Dppa2 gene, suggesting Dppa2 might be under transcriptional regulation of Oct4. Further chromatin immunoprecipitation (ChIP) assay and electrophoretic gel mobility shift assay (EMSA) assay confirmed that we for the first time determined-1964--1950bp region downstream of Dppa2 is the recognition site of Oct4 transcription factor.
In summary, our study indicated that Dppa2 play a role in maintaining mESCs undifferentiation. Besides, we for the first time analyzed the TSS of Dppa2, isolated Dppa2 promoter, confirmed Dppa2 receives a (positive) transcriptional regulation from Oct4 and it might play as a downstream gene of Oct4 in maintaining mESCs undifferentiation condition. Our study laid a foundation for a further clarification of molecular regulation mechanisms in maintaining ESCs undifferentiation.
我们首先利用Real-time PCR分析了Dppa2基因在mESCs分化过程中的表达水平变化情况,并将之与Oct4, Sox2和Nanog基因的表达变化情况进行了分析和比较。我们采用了两种使mESCs分化的方法：悬浮培养形成拟胚体(Embryoid bodies, EBs)和维甲酸(A11trans-retinoic acid, atRA)诱导mESCs分化。我们的结果表明,在两种分化条件下,Dppa2基因均表现出和Oct4, Sox2或Nanog基因相似的表达变化趋势,在分化早期时间点均出现不同程度的表达上调,而在mESCs分化后期各基因表达水平均显著下调至非常低的水平。
为了获得理想的mESCs转染效率,实现载体法对Dppa2基因的RNA干扰(RNA interference, RNAi),我们对脂质体LipofectamineTM2000转染mESCs的条件进行了摸索和优化。结果提示低密度培养条件下(30-50%)的mESCs更容易获得较高的转染效率(≥50%)。
利用RNAi技术我们首次对mESCs中Dppa2基因进行了表达抑制。RT-PCR和Real-time PCR结果表明我们的RNAi是成功的,两个针对Dppa2基因的干扰组中干扰效率分别达到了85%(sh1)和78%(sh2)。Dppa2基因的表达被抑制后mESCs表现出了一定的分化趋势,表现为：碱性磷酸酶(Alkaline phosphatase, AKP)活性下降,染色有所减弱；多能性标记基因Oct4和Nanog的表达水平出现了微弱的下调(下调约20%左右)以及部分胚层标志基因(如内胚层的Fst和滋养外胚层的Psx1基因)表达出现不同水平的上调。此外,通过BrdU嵌入实验对细胞的增殖能力变化进行检测,结果表明,Dppa2基因的表达抑制引起了mESCs增殖能力明显下降。端粒酶活性检测结果显示Dppa2基因的表达抑制并未引起mESCs端粒酶活性的明显改变。
利用5'-RACE技术,我们将Dppa2基因的转录起始位点(Transcriptional start site, TSS)确定在其ATG上游-30bp处。此外,利用双荧光素酶报告系统,我们首次分析和检测了Dppa2基因不同启动子候选区段的活性,结果提示-176bp-+293bp区域为Dppa2核心启动子所在区。Dppa2启动子在mESCs中具有高启动活性,在P19细胞(小鼠胚胎癌细胞系,mECCs)中有一定启动活性,而在我们目前所检测的其它几种细胞中均基本无启动活性,提示Dppa2启动子在多能性细胞中特异表达。
通过生物信息学分析我们发现在Dppa2基因上游存在多个Oct4转录因子保守识别位点(ATGC(A/T)(A/T)-(A/T)),提示该基因可能受到Oct4的转录调控。进一步通过染色质免疫共沉淀(chromatinimmunoprecipitation, ChIP)和凝胶电泳迁移率变动分析(electrophoretic gel mobility shift assay, EMSA)等实验验证,我们首次确定了Dppa2基因上游-1964--1950处为Oct4转录调控识别位点。
总之,我们的研究结果表明Dppa2基因在维持mESCs不分化过程中发挥着一定的作用。此外,我们首次通过实验分析了该基因的转录起始位点并分离得到其启动子,并证实该基因受到了Oct4的(正)转录调控,提示Dppa2基因可能通过作为Oct4的下游基因之一参与维持mESCs不分化状态。我们的结果为进一步阐明维持胚胎干细胞不分化状态的分子调控机制奠定良好的基础。
Dppa2 (Developmental pluripotency-associated 2) is a recently identified gene specifically expressed in pluripotent cells. Current information on DPPA2 (human)/Dppa2 (mouse) were collected from the expression analysis of DPPA2/Dppa2 as a pluripotent marker and DPPA2/Dppa2 expression in cancer cells as a cancer/testis gene. Despite the current information, a further study in DPPA2/Dppa2 function as well as the mechanisms is needed. Our study is aimed at exploring the role of Dppa2 in maintaining the mESCs undifferentiation and the possible mechanisms.
Firstly, we used Real-time PCR to analyze the change of Dppa2 expression during mESCs differentiation, and compared the change with those of Oct4, Sox2 and Nanog. We adapted two methods to induce mESCs differentiation:suspension culture to form embryoid bodies (EBs) and all trans-retinoic acid (atRA) induction. Our results showed that, under the two differentiation conditions, Dppa2 displayed a similar trend of expression change to Oct4, Sox2 and Nanog:The expression was upregulated in earlier time then significantly downregulated to a very low level in later period during mESCs differentiation.
To achieve ideal transfection efficiency on mESCs and a successful RNA interference (RNAi) on Dppa2, we investigated and optimized the conditions of mESCs transfection with LipofectamineTM2000. Our results suggest that a low confluence of mESCs (30-50%) is prone to result in a relatively high transfection efficiency (≥50%).
For the first time, we used RNAi to inhibit Dppa2 expression in mESCs. The results of RT-PCR and Real-time PCR demonstrated that the RNAi on Dppa2 is successful, where the interference efficiencies in two inference groups shl and sh2 respectively reached 85% and 78%. After Dppa2 RNAi, mESCs showed considerable trends of differentiation, which indicated as a decreased AKP activity, a weak stain; slight downregulation (-20%) of Oct4 and Nanog expression as well as differently levels of upregulation on parts of germ layer marker genes (such as Fst of endoderm and Psx1 of Trophoblastic ectoderm). In addition, we detected the change of mESCs proliferation capability using BrdU assay. The results indicated that the inhibition of Dppa2 expression caused a significant decline on mESCs'proliferation capability.
We located the transcriptional start site (TSS) of Dppa2 at-30bp upstream its start codon ATG using 5'-RACE method. In addition, we used a dual luciferase reporter system to analyze and detect the activities of different candidate regions of Dppa2 promoter. The results suggested that-176bp-+293bp region is the basal promoter region of Dppa2. Dppa2 promoter has a high activity in mESCs and a certain activity in P19 (mouse embryo carcinoma cells, mECCs) while nearly no activity in other cell lines we have detected, suggesting the promoter has a specific activity in pluripotent cells.
We performed bioinformatics analysis and found that multiple recognition sites (ATGC(A/T)(A/T)-(A/T)) of Oct4 transcription factor exist in upstream region of Dppa2 gene, suggesting Dppa2 might be under transcriptional regulation of Oct4. Further chromatin immunoprecipitation (ChIP) assay and electrophoretic gel mobility shift assay (EMSA) assay confirmed that we for the first time determined-1964--1950bp region downstream of Dppa2 is the recognition site of Oct4 transcription factor.
In summary, our study indicated that Dppa2 play a role in maintaining mESCs undifferentiation. Besides, we for the first time analyzed the TSS of Dppa2, isolated Dppa2 promoter, confirmed Dppa2 receives a (positive) transcriptional regulation from Oct4 and it might play as a downstream gene of Oct4 in maintaining mESCs undifferentiation condition. Our study laid a foundation for a further clarification of molecular regulation mechanisms in maintaining ESCs undifferentiation.
引文
[1]Smith AG. Embryo-derived stem cells:of mice and men. Annu Rev Cell Dev Biol, 2001,17:435-462.
[2]Keller G. Embryonic stem cell differentiation:Emergence of a new era in biology and medicine. Genes Dev,2005,19 (10):1129-1155.
[3]Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell,1998,95:379-391.
[4]Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell,2003,113: 631-642.
[5]Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell,2003,113:643-655.
[6]Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev,2003,17:126-140.
[7]Zhang JQ, Tam WL, Tong GQ, Wu Q, Chan HY, Soh BS, Lou Y, Yang J, Ma Y, Chai L, Ng HH, Lufkin T, Robson P, Lim B. Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5fl. Nat Cell Biol,2006,8:1114-1123.
[8]Tsuneyoshi N, Sumi T, Onda H, Nojima H, Nakatsuji N, Suemori H. PRDM14 suppresses expression of differentiation marker genes in human embryonic stem cells. Biochem Biophys Res Commun,2008,367:899-905.
[9]Wang ZX, Kueh JLL, Teh CHL, Rossbach M, Lim L, Li P, Wong KY, Lufkin T, Robson P, Stanton LW. Zfp206 is a transcription factor that controls pluripotency of embryonic stem cells. Stem Cells,2007,25:2173-2182.
[10]Du J, Lin G, Nie ZY, Lu GX. Molecular cloning and characterization analysis of HPESCRG1, a novel gene expressed specifically in human embryonic stem cell. Zhonghua Yi Xue Yi Chuan Xue Za Zhi,2004,6:542-547.
[11]Bortvin A, Eggan K, Skaletsky H, Akutsu H, Berry DL, Yanagimachi R, Page DC, Jaenisch R. Incomplete reactivation of Oct4-related genes in mouse embryos cloned form somatic nuclei. Development,2003,130:1673-1680.
[12]Maldonado-Saldivia J, Bergen J, Krouskos M, Gilchrist M, Lee C, Li R, Sinclair AH, Surani MA, Western PS. Dppa2 and Dppa4 are closely linked SAP motif genes restricted to pluripotent cells and the germ line. Stem Cells,2007,25: 19-28.
[13]Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell,2007,131:1-12.
[14]John T, Caballero OL, Svobodova SJ, Kong A, Chua R, Browning J, Fortunato S, Deb S, Hsu M, Gedye CA, Davis ID, Altorki N, Simpson AJ, Chen YT, Monk M, Cebon JS. ESCA/DPPA2 is an embryo-cancer antigen that is coexpressed with cancer-testis antigens in non-small cell lung cancer. Clin.Cancer Res.,2008, 14:3291-3298.
[15]Monk M, Hitchins M, Hawes S. Differential expression of the embryo/cancer gene ESCA (DPPA2), the cancer/testis gene BORIS and the pluripotency structural gene OCT4, in human preimplantation development. Mol. Hum. Reprod.,2008,14:347-355.
[16]Chew JL, Loh YH, Zhang WS, et al. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol,2005,25 (14):6031-6046.
[17]Pfeifer A, Ikawa M, Dayn Y, et al. Transgenesis by lentiviral vectors:lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos [J]. Proc Natl Acad Sci USA,2002,99 (4):2140-2145.
[18]Zhang MY, Li H, Dong WW, et al. Establishment of a mouse embryonic stem cell line integrated with pTet-on gene. Acta Lab Anim Sci Sin,2006,14 (3): 161-166.
[19]Velkey JM, O'Shea KS. Oct4 RNA interference induces trophectoderm differentiation in mouse embryonic stem cells. Genesis,2003,37:18-24.
[20]Hay DC, Sutherland L, Clark J, Burdon T. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells,2004,22:225-235.
[21]Hough SR, Clements I, Welch PJ, Wiederholt KA. Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of Oct4 and Nanog. Stem Cells,2006,24:1467-1475
[22]Hyslop L, Stojkovic M, Armstrong L, Walter T, Stojkovic P, Przyborski S, Herbert M, Murdoch A, Strachan T, Lako M. Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages. Stem Cells,2005,23,1035-1043.
[23]Sharov AA, Masui S, Sharova LV, Piao Y, Aiba K, Matoba R, Xin L, Niwa H., Ko MS. Identification of Pou5fl, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data. BMC Genomics,2008,9:269-287.
[24]Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell,2005,122:947-956.
[25]Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet,2006,38:431-440.
[1]Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Narture,1998,391:806-811.
[2]Hayafune M, Miyano-Kurosaki N, Takaku H, Park WS. Silencing of HIV-1 gene expression by siRNAs in transduced cells. Nucleosides Nucleotides Nucleic Acids, 2006,25(7):795-799.
[3]Zamecnik PC & Stephensen ML Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci., 1978,75:280.
[4]Guo S. & Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, 1995,81:611.
[5]Zamore PD, Tuschl T, Sharp PA, and Bartel DP. RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell,2000,101,25.
[6]Elbashire SM, Lendeckel W & Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev.,2001b,15:188.
[7]Mello CC & Conte Jr D. Revealing the world of RNA interference. Nature,2004, 431(7006):338-342.
[8]Kumar R, Conklin DS & Mittal V. High-throughput selection of effective RNAi probes for gene silencing. Genome Res,2003,13(10):2333-2340.
[9]Hsieh AC, Bo R, Manola J, Vazquez F, Bare O & Khvorova A, et al. A library of SiRNA duplexes targeting the phosphoinositide 3-kinase pathway:determinants of gene silencing for use in cell-based screens. Nucleic Acids Res,2004,32(3):893-901.
[10]Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.
Proc Natl Acad Sci USA,2001,98(17):9742-9747.
[11]Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature,2001,411(6836):494-498.
[12]Kao SC., Krichevsky AM, Kosik KS, Tsai LH. BACE1 suppression by RNA interference in primary cortical neurons. JBiol Chem,2004,279(3),1942-1949.
[13]Krick S, Eul BG, Hanze J, Savai R, Grimminger F, Seeger W, et al. Role of HIF-1{Alpha} in hypoxia-induced apoptosis of primary alveolar epithelial type II cells. Am J Respir Cell Mol Biol,2005,32(5):395-403.
[14]Ikeda R, Yoshida K, Tsukahara S, Sakamoto Y, Tanaka H, Furukawa KI, et al. PLZF promotes osteoblastic differentiation of human mesenchymal stem cells as an upstream regulator of CBFA1. JBiol Chem,2004,280(9):8523-8530.
[15]Omi K, Tokunaga K, Hohjoh H. Long-lasting RNAi activity in mammalian neurons. FEBS Lett,2004,558(1-3):89-95.
[16]Tuschl T. Expanding small RNA interference. Nat Biotechnol,2002,20(5):446-448.
[17]Miyagishi M & Taira K. Strategies for generation of an SiRNA expression library directed against the human genome. Oligonucleotides,2003,13(5):325-333.
[18]Siolas D, Lerner C, Burchard J, Ge W, Linsley PS, Paddison PJ, et al. Synthetic ShRNAs as potent RNAi triggers. Nat Biotechnol,2005,23(2):227-231.
[19]Brummelkamp T R, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell,2002,2(3):243-247.
[20]Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet,2003,33(3):401-406.
[21]Arts GJ, Langemeijer E, Tissingh R, Ma L, Pavliska H, Dokic K, et al. Adenoviral vectors expressing SiRNAs for discovery and validation of gene function. Genome Res,2003,13(10):2325-2332.
[22]Hemann MT, Fridman JS, Zilfou JT, Hernando E, Paddison PJ, Cordon-Cardo C, et al. An Epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor pheno-types in vivo. Nat Genet,2003,33(3):396-400.
[23]Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet,2003,33(3):401-406.
[24]Kunath T, Gish G, Lickert H, Jones N, Pawson T, Rossant J. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nat Biotechnol,2003,21(5):559-561.
[25]Chen J, Wall NR, Kocher K, Duclos N, Fabbro D, Neuberg D, et al. Stable expression of small interfering RNA sensitizes TEL-PDGFbetaR to inhibition with imatinib or rapamycin. J Clin Invest,2004,113(12):1784-1791.
[26]Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell,2002,2(3):243-247.
[27]Lapteva N, Yang AG, Sanders DE, Strube RW, Chen SY CXCR4 knockdown by small interfering RNA abrogates breast tumor growth in vivo. Cancer Gene Ther 2004,12(1):84-89.
[28]Karasarides M, Chiloeches A, Hayward R, Niculescu-Duvaz D, Scanlon I, Friedlos F, et al. B-RAF is a therapeutic target in melanoma. Oncogene,2004, 23(37):6292-6298.
[29]Verma UN, Surabhi RM, Schmaltieg A, Becerra C, Gaynor RB. Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clin Cancer Res,2003,9(4),1291-1300.
[30]Yang G, Thompson JA, Fang B, Liu J. Silencing of H-ras gene expression by retrovirus-mediated SiRNA decreases transformation efficiency and tumor growth in a model of human ovarian cancer. Oncogene,2003,22(36):5694-5701.
[31]Dave RS & Pomerantz RJ. Antiviral effects of human immunodeficiency virus type 1-specific small interfering RNAs against targets conserved in select neurotropic viral strains. J Virol,2004,78(24):13687-13696.
[32]Wilson JA, Jayasena S, Khvorova A, Sabatinos S, Rodrigue-Gervais IG, Arya S, et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc Natl Acad Sci U S A,2003, 100(5):2783-2788.
[33]Zhang Y, Li T, Fu L, Yu C, Li Y, Xu X, et al. Silencing SARS-CoV spike protein expression in cultured cells by RNA interference. FEBS Lett,2004,560(1-3): 141-146.
[34]Qin XF, An DS, Chen IS, Baltimore D. Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A,2003,100(1):183-188.
[35]Li MJ, Bauer G, Michienzi A, Yee JK, Lee NS, Kim J, et al. Inhibition of HIV-1 infection by lentiviral vectors expressing pol Ⅲ-promoted anti-HIV RNAs. Mol Ther,2003,8(2):196-206.
[36]Randall G, Grakoui A, Rice CM. Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs. Proc Natl Acad Sci U S A,2003,100(1):235-240.
[37]Randall G & Rice CM. Interfering With hepatitis C virus RNA replication. Virus Res,2004,102(1):19-25.
[38]Giladi H, Ketzinel-Gilad M, Rivkin L, Felig Y, Nussbaum O, Galun E. Small interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther,2003, 8(5):769-776.
[39]Yang ZG, Chen Z, Ni Q, Xu N, Shao JB, Yao HP. Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro. World J Gastroenterol, 2005,11(4):498-502.
[40]Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery of synthetic SiRNAs in adult mice. JMol Biol,2003,327(4):761-766.
[41]Kumar R, Adams B, Oldenburg A, Musiyenko A, Barik S. Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falcipa-rum:demonstration of its essential role using RNA interference. Malar J,2002,1(1):5.
[42]Shao MX, Nakanaga T, Nadel JA. Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells. Am J Physiol Lung Cell Mol Physiol,2004, 287(2):L420-L427.
[43]Jeffery TK, Upton PD, Trembath RC, Morrell NW. BMP4 inhibits proliferation and promotes myocyte differentiation of lung fibroblasts via Smadl and JNK pathways. Am J Physiol Lung Cell Mol Physiol,2005,288(2):L370-L378.
[44]Finigan JH, Dudek SM, Singleton PA, Chiang ET, Jacobson JR, Camp SM, et al. Activated protein C mediates novel lung endothelial barrier enhancement:role of sphingosine 1-phosphate receptor transactivation. J Biol Chem,2005,280(17): 17286-17293.
[45]Ulanova M, Puttagunta L, Marcet-Palacios M, Duszyk M, Steinhoff U, Duta F, et al. Syk tyrosine kinase participates in betal-integrin signaling and inflammatory responses in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol,2005, 288(3):L497-L507.
[46]Gou D, Narasaraju T, Chintagari NR, Jin N, Wang P, Liu L. Gene silencing in alveolar type II cells using cell-specific promoter in vitro and in vivo. Nucleic Acids Res,2004,32(17):e134.
[47]Braasch DA, Paroo Z, Constantinescu A, Ren G, Oz OK, Mason RP, et al. Biodistribution of phosphodiester and phosphorothioate SiRNA. Bioorg Med Chem Lett,2004,14(5),1139-1143.
[48]Bertrand JR, Pottier M, Vekris A, Opolon P, Maksimenko A, Malvy C. Comparison of antisense oligonucleotides and SiRNAs in cell culture and in vivo. Biochem Biophys Res Commun,2002,296(4):1000-1004.
[49]Paroo Z & Corey DR. Challenges for RNAi in Vivo. Trends Biotechnol,2004, 22(8):390-394.
[50]Dykxhoorn DM & Lieberman J. The silent revolution:RNA interference as basic biology, research tool, and therapeutic. Annu Rev Med,2005,56:401-423.
[51]Hall AH, Wan J, Shaughnessy EE, Ramsay SB, Alexander KA. RNA interference using boranophosphate SiRNAs:structure-activity relationships. Nucleic Acids Res,2004,32(20):5991-6000.
[52]Elmen J, Thonberg H, Ljungberg K, Frieden M, Westergaard M, Xu Y, et al. Locked nucleic acid (LNA) mediated improve-ments in SiRNA stability and functionality. Nucleic Acids Res,2005,33(1):439-447.
[53]Allerson CR, Sioufi N, Jarres R, Prakash TP, Naik N, Berdeja A, et al. Fully 2V-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA. J Med Chem,2005, 48(4):901-904.
[54]Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed SiRNA in vivo. Gene Ther,2005,12(5):461-466.
[55]Minakuchi Y, Takeshita F, Kosaka N, Sasaki H, Yamamoto Y, Kouno M, et al. Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo. Nucleic Acids Res,2004,32(13):e109.
[56]Khan A, Benboubetra M, Sayyed PZ, Ng KW, Fox S, Beck G, et al. Sustained polymeric delivery of gene silencing antisense ODNs, SiRNA, DNAzymes and ribozymes:in vitro and in vivo studies. J Drug Target,2004,12(6):393-404.
[57]Lewis DL & Wolff JA. Delivery of SiRNA and SiRNA expression constructs to adult mammals by hydrodynamic intravascular injection. Methods Enzymol,2005, 392:336-350.
[58]Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther,1999,6(7):1258-1266.
[59]Ge Q, Filip L, Bai A, Nguyen T, Eisen HN, Chen J. Inhibition of influenza virus production in virus-infected mice by RNA interference. Proc Natl Acad Sci U S A, 2004,101(23):8676-8681.
[60]Hassani Z, Lemkine GF, Erbacher P, Palmier K, Alfama G, Giovannangeli C, et al. Lipid-mediated SiRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels. J Gene Med,2004,7(2):198-207.
[61]Zhang Y, Zhang YF, Bryant J, Charles A, Boado RJ, Pardridge WM. Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intra-cranial brain cancer. Clin Cancer Res,2004, 10(11):3667-3677.
[62]Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed SiRNA in vivo. Gene Ther,2005,12(5):461-466.
[63]Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G, et al. Cancer SiRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res,2004,32(19):e149.
[64]Lorenz C, Hadwiger P, John M, Vornlocher HP, Unverzagt C. Steroid and lipid conjugates of SiRNAs to enhance cellular uptake and gene silencing in liver cells. Bioorg Med Chem Lett,2004,14(19):4975-4977.
[65]Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R, Donoghue M, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified SiRNAs. Nature,2004,432(7014):173-178.
[66]Kim B, Tang Q, Biswas PS, Xu J, Schiffelers RM, Xie FY, et al. Inhibition of ocular angiogenesis by SiRNA targeting vascular endothelial growth factor pathway genes:thera-peutic strategy for herpetic stromal keratitis. Am J Pathol, 2004,165(6):2177-2185.
[67]Takabatake Y, Isaka Y, Mizui M, Kawachi H, Shimizu F, Ito T, et al. (in press). Exploring RNA interference as a therapeutic strategy for renal disease. Gene Ther.
[68]Akaneya Y, Jiang B, Tsumoto T. RNAi-induced gene silencing by local electroporation in targeting brain region. JNeuro-physio,2005,193(1):594-602.
[69]Matsuda T & Cepko CL. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci U S A,2004,101(1):16-22.
[70]Golzio M, Mazzolini L, Moller P, Rols MP, Teissie J. Inhibition of gene expression in mice muscle by in vivo electrically mediated SiRNA delivery. Gene Ther,2005,12(3):246-251.
[71]Zhang L, Nolan E, Kreitschitz S, Rabussay DP. Enhanced delivery of naked DNA to the skin by non-invasive in vivo electro-poration. Biochim Biophys Acta, 2002,1572(1):1-9.
[72]Jiang M, Rubbi CP, Milner J. Gel-based application of SiRNA to human
epithelial cancer cells induces RNAi-dependent apoptosis. Oligonucleotides,2004, 14(4):239-248.
[73]Kim TW, Lee JH, He L, Boyd DA, Hardwick JM, Hung CF, et al. Modification of professional antigen-presenting cells with small interfering rna in vivo to enhance cancer vaccine potency. Cancer Res,2005,65(1):309-316.
[74]Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med,2003, 9(12):1539-1544.
[75]Xia H, Mao Q, Eliason SL, Harper SQ, Martins IH, Orr HT, et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med,2004,10(8):816-820.
[76]Banerjea A, Li MJ, Bauer G, Remling L, Lee NS, Rossi J, et al. Inhibition of HIV-1 by lentiviral vector-transduced SiRNAs in T lymphocytes differentiated in SCID-Hu mice and CD34+ progenitor cell-derived macrophages. Mol Ther,2003, 8(1):62-71.
[77]Hassani Z, Lemkine GF, Erbacher P, Palmier K, Alfama G, Giovannangeli C, et al. Lipid-mediated SiRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels. J Gene Med,2004,7(2):198-207.
[78]Marshall E. Gene therapy. Second child in french trial is found to have leukemia. Science,2003,299(5605):320.
[79]Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol, 2003,21(6):635-637.
[80]Couzin J. Molecular biology. RNAi shows cracks in its armor. Science,2004, 306(5699):1124-1125.
[81]Jackson A L & Linsley PS. Noise amidst the silence:off-target effects of SiRNAs? Trends Genet,2004,20(11):521-524.
[82]Scacheri PC, Rozenblatt-Rosen O, Caplen NJ. Wolfsberg TG, Umayam L, Lee JC, et al. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc Natl Acad Sci U S A,2004 101(7):1892-1897.
[83]Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA,2002,99(9):6047-6052.
[84]Pusch O, Boden D, Silbermann R, Lee F, Tucker L, Ramratnam B. Nucleotide sequence homology requirements of HIV-1-specific short hairpin RNA. Nucleic Acids Res,2003,31(22):6444-6449.
[85]Vickers TA, Koo S, Bennett CF, Crooke ST, Dean NM, Baker BF. Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. JBiol Chem,2003,278(9):7108-7118.
[86]Saxena S, Jonsson ZO, Dutta A. Small RNAs with imperfect match to endogenous MRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. J Biol Chem,2003,278(45):44312-44319.
[87]Holen T, Amarzguioui M, Babaie E, Prydz H. Similar behaviour of single-strand and double-strand SiRNAs suggests they act through a common RNAi pathway. Nucleic Acids Res,2003,31(9):2401-2407.
[88]Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell,2003,115(2):199-208.
[89]Snove OJr & Holen T. Many commonly used SiRNAs risk off-target activity. Biochem Biophys Res Commun,2004,319(1):256-263.
[90]Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol,2003,5(9):834-839.
[91]Kariko K, Bhuyan P, Capodici J, Ni H, Lubinski J, Friedman H, et al. Exogenous SiRNA mediates sequence-independent gene suppression by signaling through toll-like receptor 3. Cells Tissues Organs,2004,177(3):132-138.
[92]Zhou HS, Liu DP, Liang CC. Challenges and strategies:the immune responses in gene therapy. Med Res Rev,2004,24(6):748-761.
[2]Keller G. Embryonic stem cell differentiation:Emergence of a new era in biology and medicine. Genes Dev,2005,19 (10):1129-1155.
[3]Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell,1998,95:379-391.
[4]Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell,2003,113: 631-642.
[5]Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell,2003,113:643-655.
[6]Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev,2003,17:126-140.
[7]Zhang JQ, Tam WL, Tong GQ, Wu Q, Chan HY, Soh BS, Lou Y, Yang J, Ma Y, Chai L, Ng HH, Lufkin T, Robson P, Lim B. Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5fl. Nat Cell Biol,2006,8:1114-1123.
[8]Tsuneyoshi N, Sumi T, Onda H, Nojima H, Nakatsuji N, Suemori H. PRDM14 suppresses expression of differentiation marker genes in human embryonic stem cells. Biochem Biophys Res Commun,2008,367:899-905.
[9]Wang ZX, Kueh JLL, Teh CHL, Rossbach M, Lim L, Li P, Wong KY, Lufkin T, Robson P, Stanton LW. Zfp206 is a transcription factor that controls pluripotency of embryonic stem cells. Stem Cells,2007,25:2173-2182.
[10]Du J, Lin G, Nie ZY, Lu GX. Molecular cloning and characterization analysis of HPESCRG1, a novel gene expressed specifically in human embryonic stem cell. Zhonghua Yi Xue Yi Chuan Xue Za Zhi,2004,6:542-547.
[11]Bortvin A, Eggan K, Skaletsky H, Akutsu H, Berry DL, Yanagimachi R, Page DC, Jaenisch R. Incomplete reactivation of Oct4-related genes in mouse embryos cloned form somatic nuclei. Development,2003,130:1673-1680.
[12]Maldonado-Saldivia J, Bergen J, Krouskos M, Gilchrist M, Lee C, Li R, Sinclair AH, Surani MA, Western PS. Dppa2 and Dppa4 are closely linked SAP motif genes restricted to pluripotent cells and the germ line. Stem Cells,2007,25: 19-28.
[13]Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell,2007,131:1-12.
[14]John T, Caballero OL, Svobodova SJ, Kong A, Chua R, Browning J, Fortunato S, Deb S, Hsu M, Gedye CA, Davis ID, Altorki N, Simpson AJ, Chen YT, Monk M, Cebon JS. ESCA/DPPA2 is an embryo-cancer antigen that is coexpressed with cancer-testis antigens in non-small cell lung cancer. Clin.Cancer Res.,2008, 14:3291-3298.
[15]Monk M, Hitchins M, Hawes S. Differential expression of the embryo/cancer gene ESCA (DPPA2), the cancer/testis gene BORIS and the pluripotency structural gene OCT4, in human preimplantation development. Mol. Hum. Reprod.,2008,14:347-355.
[16]Chew JL, Loh YH, Zhang WS, et al. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol,2005,25 (14):6031-6046.
[17]Pfeifer A, Ikawa M, Dayn Y, et al. Transgenesis by lentiviral vectors:lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos [J]. Proc Natl Acad Sci USA,2002,99 (4):2140-2145.
[18]Zhang MY, Li H, Dong WW, et al. Establishment of a mouse embryonic stem cell line integrated with pTet-on gene. Acta Lab Anim Sci Sin,2006,14 (3): 161-166.
[19]Velkey JM, O'Shea KS. Oct4 RNA interference induces trophectoderm differentiation in mouse embryonic stem cells. Genesis,2003,37:18-24.
[20]Hay DC, Sutherland L, Clark J, Burdon T. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells,2004,22:225-235.
[21]Hough SR, Clements I, Welch PJ, Wiederholt KA. Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of Oct4 and Nanog. Stem Cells,2006,24:1467-1475
[22]Hyslop L, Stojkovic M, Armstrong L, Walter T, Stojkovic P, Przyborski S, Herbert M, Murdoch A, Strachan T, Lako M. Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages. Stem Cells,2005,23,1035-1043.
[23]Sharov AA, Masui S, Sharova LV, Piao Y, Aiba K, Matoba R, Xin L, Niwa H., Ko MS. Identification of Pou5fl, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data. BMC Genomics,2008,9:269-287.
[24]Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell,2005,122:947-956.
[25]Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet,2006,38:431-440.
[1]Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Narture,1998,391:806-811.
[2]Hayafune M, Miyano-Kurosaki N, Takaku H, Park WS. Silencing of HIV-1 gene expression by siRNAs in transduced cells. Nucleosides Nucleotides Nucleic Acids, 2006,25(7):795-799.
[3]Zamecnik PC & Stephensen ML Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci., 1978,75:280.
[4]Guo S. & Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, 1995,81:611.
[5]Zamore PD, Tuschl T, Sharp PA, and Bartel DP. RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell,2000,101,25.
[6]Elbashire SM, Lendeckel W & Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev.,2001b,15:188.
[7]Mello CC & Conte Jr D. Revealing the world of RNA interference. Nature,2004, 431(7006):338-342.
[8]Kumar R, Conklin DS & Mittal V. High-throughput selection of effective RNAi probes for gene silencing. Genome Res,2003,13(10):2333-2340.
[9]Hsieh AC, Bo R, Manola J, Vazquez F, Bare O & Khvorova A, et al. A library of SiRNA duplexes targeting the phosphoinositide 3-kinase pathway:determinants of gene silencing for use in cell-based screens. Nucleic Acids Res,2004,32(3):893-901.
[10]Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.
Proc Natl Acad Sci USA,2001,98(17):9742-9747.
[11]Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature,2001,411(6836):494-498.
[12]Kao SC., Krichevsky AM, Kosik KS, Tsai LH. BACE1 suppression by RNA interference in primary cortical neurons. JBiol Chem,2004,279(3),1942-1949.
[13]Krick S, Eul BG, Hanze J, Savai R, Grimminger F, Seeger W, et al. Role of HIF-1{Alpha} in hypoxia-induced apoptosis of primary alveolar epithelial type II cells. Am J Respir Cell Mol Biol,2005,32(5):395-403.
[14]Ikeda R, Yoshida K, Tsukahara S, Sakamoto Y, Tanaka H, Furukawa KI, et al. PLZF promotes osteoblastic differentiation of human mesenchymal stem cells as an upstream regulator of CBFA1. JBiol Chem,2004,280(9):8523-8530.
[15]Omi K, Tokunaga K, Hohjoh H. Long-lasting RNAi activity in mammalian neurons. FEBS Lett,2004,558(1-3):89-95.
[16]Tuschl T. Expanding small RNA interference. Nat Biotechnol,2002,20(5):446-448.
[17]Miyagishi M & Taira K. Strategies for generation of an SiRNA expression library directed against the human genome. Oligonucleotides,2003,13(5):325-333.
[18]Siolas D, Lerner C, Burchard J, Ge W, Linsley PS, Paddison PJ, et al. Synthetic ShRNAs as potent RNAi triggers. Nat Biotechnol,2005,23(2):227-231.
[19]Brummelkamp T R, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell,2002,2(3):243-247.
[20]Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet,2003,33(3):401-406.
[21]Arts GJ, Langemeijer E, Tissingh R, Ma L, Pavliska H, Dokic K, et al. Adenoviral vectors expressing SiRNAs for discovery and validation of gene function. Genome Res,2003,13(10):2325-2332.
[22]Hemann MT, Fridman JS, Zilfou JT, Hernando E, Paddison PJ, Cordon-Cardo C, et al. An Epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor pheno-types in vivo. Nat Genet,2003,33(3):396-400.
[23]Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet,2003,33(3):401-406.
[24]Kunath T, Gish G, Lickert H, Jones N, Pawson T, Rossant J. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nat Biotechnol,2003,21(5):559-561.
[25]Chen J, Wall NR, Kocher K, Duclos N, Fabbro D, Neuberg D, et al. Stable expression of small interfering RNA sensitizes TEL-PDGFbetaR to inhibition with imatinib or rapamycin. J Clin Invest,2004,113(12):1784-1791.
[26]Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell,2002,2(3):243-247.
[27]Lapteva N, Yang AG, Sanders DE, Strube RW, Chen SY CXCR4 knockdown by small interfering RNA abrogates breast tumor growth in vivo. Cancer Gene Ther 2004,12(1):84-89.
[28]Karasarides M, Chiloeches A, Hayward R, Niculescu-Duvaz D, Scanlon I, Friedlos F, et al. B-RAF is a therapeutic target in melanoma. Oncogene,2004, 23(37):6292-6298.
[29]Verma UN, Surabhi RM, Schmaltieg A, Becerra C, Gaynor RB. Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clin Cancer Res,2003,9(4),1291-1300.
[30]Yang G, Thompson JA, Fang B, Liu J. Silencing of H-ras gene expression by retrovirus-mediated SiRNA decreases transformation efficiency and tumor growth in a model of human ovarian cancer. Oncogene,2003,22(36):5694-5701.
[31]Dave RS & Pomerantz RJ. Antiviral effects of human immunodeficiency virus type 1-specific small interfering RNAs against targets conserved in select neurotropic viral strains. J Virol,2004,78(24):13687-13696.
[32]Wilson JA, Jayasena S, Khvorova A, Sabatinos S, Rodrigue-Gervais IG, Arya S, et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc Natl Acad Sci U S A,2003, 100(5):2783-2788.
[33]Zhang Y, Li T, Fu L, Yu C, Li Y, Xu X, et al. Silencing SARS-CoV spike protein expression in cultured cells by RNA interference. FEBS Lett,2004,560(1-3): 141-146.
[34]Qin XF, An DS, Chen IS, Baltimore D. Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A,2003,100(1):183-188.
[35]Li MJ, Bauer G, Michienzi A, Yee JK, Lee NS, Kim J, et al. Inhibition of HIV-1 infection by lentiviral vectors expressing pol Ⅲ-promoted anti-HIV RNAs. Mol Ther,2003,8(2):196-206.
[36]Randall G, Grakoui A, Rice CM. Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs. Proc Natl Acad Sci U S A,2003,100(1):235-240.
[37]Randall G & Rice CM. Interfering With hepatitis C virus RNA replication. Virus Res,2004,102(1):19-25.
[38]Giladi H, Ketzinel-Gilad M, Rivkin L, Felig Y, Nussbaum O, Galun E. Small interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther,2003, 8(5):769-776.
[39]Yang ZG, Chen Z, Ni Q, Xu N, Shao JB, Yao HP. Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro. World J Gastroenterol, 2005,11(4):498-502.
[40]Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery of synthetic SiRNAs in adult mice. JMol Biol,2003,327(4):761-766.
[41]Kumar R, Adams B, Oldenburg A, Musiyenko A, Barik S. Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falcipa-rum:demonstration of its essential role using RNA interference. Malar J,2002,1(1):5.
[42]Shao MX, Nakanaga T, Nadel JA. Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells. Am J Physiol Lung Cell Mol Physiol,2004, 287(2):L420-L427.
[43]Jeffery TK, Upton PD, Trembath RC, Morrell NW. BMP4 inhibits proliferation and promotes myocyte differentiation of lung fibroblasts via Smadl and JNK pathways. Am J Physiol Lung Cell Mol Physiol,2005,288(2):L370-L378.
[44]Finigan JH, Dudek SM, Singleton PA, Chiang ET, Jacobson JR, Camp SM, et al. Activated protein C mediates novel lung endothelial barrier enhancement:role of sphingosine 1-phosphate receptor transactivation. J Biol Chem,2005,280(17): 17286-17293.
[45]Ulanova M, Puttagunta L, Marcet-Palacios M, Duszyk M, Steinhoff U, Duta F, et al. Syk tyrosine kinase participates in betal-integrin signaling and inflammatory responses in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol,2005, 288(3):L497-L507.
[46]Gou D, Narasaraju T, Chintagari NR, Jin N, Wang P, Liu L. Gene silencing in alveolar type II cells using cell-specific promoter in vitro and in vivo. Nucleic Acids Res,2004,32(17):e134.
[47]Braasch DA, Paroo Z, Constantinescu A, Ren G, Oz OK, Mason RP, et al. Biodistribution of phosphodiester and phosphorothioate SiRNA. Bioorg Med Chem Lett,2004,14(5),1139-1143.
[48]Bertrand JR, Pottier M, Vekris A, Opolon P, Maksimenko A, Malvy C. Comparison of antisense oligonucleotides and SiRNAs in cell culture and in vivo. Biochem Biophys Res Commun,2002,296(4):1000-1004.
[49]Paroo Z & Corey DR. Challenges for RNAi in Vivo. Trends Biotechnol,2004, 22(8):390-394.
[50]Dykxhoorn DM & Lieberman J. The silent revolution:RNA interference as basic biology, research tool, and therapeutic. Annu Rev Med,2005,56:401-423.
[51]Hall AH, Wan J, Shaughnessy EE, Ramsay SB, Alexander KA. RNA interference using boranophosphate SiRNAs:structure-activity relationships. Nucleic Acids Res,2004,32(20):5991-6000.
[52]Elmen J, Thonberg H, Ljungberg K, Frieden M, Westergaard M, Xu Y, et al. Locked nucleic acid (LNA) mediated improve-ments in SiRNA stability and functionality. Nucleic Acids Res,2005,33(1):439-447.
[53]Allerson CR, Sioufi N, Jarres R, Prakash TP, Naik N, Berdeja A, et al. Fully 2V-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA. J Med Chem,2005, 48(4):901-904.
[54]Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed SiRNA in vivo. Gene Ther,2005,12(5):461-466.
[55]Minakuchi Y, Takeshita F, Kosaka N, Sasaki H, Yamamoto Y, Kouno M, et al. Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo. Nucleic Acids Res,2004,32(13):e109.
[56]Khan A, Benboubetra M, Sayyed PZ, Ng KW, Fox S, Beck G, et al. Sustained polymeric delivery of gene silencing antisense ODNs, SiRNA, DNAzymes and ribozymes:in vitro and in vivo studies. J Drug Target,2004,12(6):393-404.
[57]Lewis DL & Wolff JA. Delivery of SiRNA and SiRNA expression constructs to adult mammals by hydrodynamic intravascular injection. Methods Enzymol,2005, 392:336-350.
[58]Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther,1999,6(7):1258-1266.
[59]Ge Q, Filip L, Bai A, Nguyen T, Eisen HN, Chen J. Inhibition of influenza virus production in virus-infected mice by RNA interference. Proc Natl Acad Sci U S A, 2004,101(23):8676-8681.
[60]Hassani Z, Lemkine GF, Erbacher P, Palmier K, Alfama G, Giovannangeli C, et al. Lipid-mediated SiRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels. J Gene Med,2004,7(2):198-207.
[61]Zhang Y, Zhang YF, Bryant J, Charles A, Boado RJ, Pardridge WM. Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intra-cranial brain cancer. Clin Cancer Res,2004, 10(11):3667-3677.
[62]Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed SiRNA in vivo. Gene Ther,2005,12(5):461-466.
[63]Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G, et al. Cancer SiRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res,2004,32(19):e149.
[64]Lorenz C, Hadwiger P, John M, Vornlocher HP, Unverzagt C. Steroid and lipid conjugates of SiRNAs to enhance cellular uptake and gene silencing in liver cells. Bioorg Med Chem Lett,2004,14(19):4975-4977.
[65]Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R, Donoghue M, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified SiRNAs. Nature,2004,432(7014):173-178.
[66]Kim B, Tang Q, Biswas PS, Xu J, Schiffelers RM, Xie FY, et al. Inhibition of ocular angiogenesis by SiRNA targeting vascular endothelial growth factor pathway genes:thera-peutic strategy for herpetic stromal keratitis. Am J Pathol, 2004,165(6):2177-2185.
[67]Takabatake Y, Isaka Y, Mizui M, Kawachi H, Shimizu F, Ito T, et al. (in press). Exploring RNA interference as a therapeutic strategy for renal disease. Gene Ther.
[68]Akaneya Y, Jiang B, Tsumoto T. RNAi-induced gene silencing by local electroporation in targeting brain region. JNeuro-physio,2005,193(1):594-602.
[69]Matsuda T & Cepko CL. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci U S A,2004,101(1):16-22.
[70]Golzio M, Mazzolini L, Moller P, Rols MP, Teissie J. Inhibition of gene expression in mice muscle by in vivo electrically mediated SiRNA delivery. Gene Ther,2005,12(3):246-251.
[71]Zhang L, Nolan E, Kreitschitz S, Rabussay DP. Enhanced delivery of naked DNA to the skin by non-invasive in vivo electro-poration. Biochim Biophys Acta, 2002,1572(1):1-9.
[72]Jiang M, Rubbi CP, Milner J. Gel-based application of SiRNA to human
epithelial cancer cells induces RNAi-dependent apoptosis. Oligonucleotides,2004, 14(4):239-248.
[73]Kim TW, Lee JH, He L, Boyd DA, Hardwick JM, Hung CF, et al. Modification of professional antigen-presenting cells with small interfering rna in vivo to enhance cancer vaccine potency. Cancer Res,2005,65(1):309-316.
[74]Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med,2003, 9(12):1539-1544.
[75]Xia H, Mao Q, Eliason SL, Harper SQ, Martins IH, Orr HT, et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med,2004,10(8):816-820.
[76]Banerjea A, Li MJ, Bauer G, Remling L, Lee NS, Rossi J, et al. Inhibition of HIV-1 by lentiviral vector-transduced SiRNAs in T lymphocytes differentiated in SCID-Hu mice and CD34+ progenitor cell-derived macrophages. Mol Ther,2003, 8(1):62-71.
[77]Hassani Z, Lemkine GF, Erbacher P, Palmier K, Alfama G, Giovannangeli C, et al. Lipid-mediated SiRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels. J Gene Med,2004,7(2):198-207.
[78]Marshall E. Gene therapy. Second child in french trial is found to have leukemia. Science,2003,299(5605):320.
[79]Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol, 2003,21(6):635-637.
[80]Couzin J. Molecular biology. RNAi shows cracks in its armor. Science,2004, 306(5699):1124-1125.
[81]Jackson A L & Linsley PS. Noise amidst the silence:off-target effects of SiRNAs? Trends Genet,2004,20(11):521-524.
[82]Scacheri PC, Rozenblatt-Rosen O, Caplen NJ. Wolfsberg TG, Umayam L, Lee JC, et al. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc Natl Acad Sci U S A,2004 101(7):1892-1897.
[83]Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA,2002,99(9):6047-6052.
[84]Pusch O, Boden D, Silbermann R, Lee F, Tucker L, Ramratnam B. Nucleotide sequence homology requirements of HIV-1-specific short hairpin RNA. Nucleic Acids Res,2003,31(22):6444-6449.
[85]Vickers TA, Koo S, Bennett CF, Crooke ST, Dean NM, Baker BF. Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. JBiol Chem,2003,278(9):7108-7118.
[86]Saxena S, Jonsson ZO, Dutta A. Small RNAs with imperfect match to endogenous MRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. J Biol Chem,2003,278(45):44312-44319.
[87]Holen T, Amarzguioui M, Babaie E, Prydz H. Similar behaviour of single-strand and double-strand SiRNAs suggests they act through a common RNAi pathway. Nucleic Acids Res,2003,31(9):2401-2407.
[88]Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell,2003,115(2):199-208.
[89]Snove OJr & Holen T. Many commonly used SiRNAs risk off-target activity. Biochem Biophys Res Commun,2004,319(1):256-263.
[90]Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol,2003,5(9):834-839.
[91]Kariko K, Bhuyan P, Capodici J, Ni H, Lubinski J, Friedman H, et al. Exogenous SiRNA mediates sequence-independent gene suppression by signaling through toll-like receptor 3. Cells Tissues Organs,2004,177(3):132-138.
[92]Zhou HS, Liu DP, Liang CC. Challenges and strategies:the immune responses in gene therapy. Med Res Rev,2004,24(6):748-761.