Blocking with double-stranded RNA of the expression of Hes5in rat bone marrow-derived neural stem cells

Abstract

Abstract: Objective To examine the efficiency of exogenous small d ouble-stranded RNA (dsRNA) in knocking down the gene expression at the post-tra nscription level, and investigate the factors that may influence the transfectio n.Method The bone marrow stromal cells of SD rat were separated and cultured i n vitro, followed by induction of the cells to evolve into neural stem cells us ing special culture medium prepared by our laboratory. Synthetic dsRNA was then transferred into the cells at varied concentrations, and the results were analy zed by Western blotting.Results The concentrations ranging from 200 to 300 nmol /L were optimal for specifically blocking the expression of Hes5, whereas the su itable concentrations for the cell survival were between 50 and 200 nmol/L.Conclusion dsRNA is capable of triggering RNA interference in neural stem cells, a nd at appropriate concentration, it may specifically and effectively knock down endogenous gene expression without sacrificing the viability of the cells.

CLC Number:

Q189

WANG Hai-yan¹, XU Ru-xiang¹, JIANG Xiao-dan¹, LUO Shen-qiu². Blocking with double-stranded RNA of the expression of Hes5in rat bone marrow-derived neural stem cells[J]. Journal of Southern Medical University, 2004, 24(01): 35-38,41.

References

[1] Savill NJ,Sherratt JA. Control of epidermal stem cell clusters by Notch-mediated lateral induction. Dev Biol[J]. 2003,258(1):141-53.
[2] Panchision DM,McKay RD. The control of neural stem cells by morphogenic signals[J]. Curr Opin Genet Dev,2002,12(4):478-87.
[3] Kageyama R,Ohtsuka T. The Notch-Hes pathway in mammalian neural development[J]. Cell Res,1999,9(3):179-88.
[4] Bryant J,Goodyear RJ,Richardson GP. Sensory organ development in the inner ear:molecular and cellular mechanisms[J].Br Med Bull,2002,63(1):39-57.
[5] Kume T,Jiang H,Topczewska JM,et al. The murine winged helix transcription factors,Foxcl and Foxc2,are both required for cardiovascular development and somitogenesis[J]. Genes Dev,2001,15(18):2470-82.
[6] Timmons L,Fire A. Specific interference by ingested dsRNA[J].Nature,1998,395(6705):854-68.
[7] Maine EM. RNAi as a tool for understanding germline development in Caenorhabditis elegans:uses and cautions[J] ]. Dev Biol,2001,239(2):177-89.
[8] Shuey DJ,McCallus DE,Giordano T. RNAi:gene-silencing in therapeutic intervention[J]. Drug Discov Today,2002,7(20):1040-6.
[9] Shi Y. Mammalian RNAi for the masses[J]. Trends Genet,2003,19(1):9-12.
[10] Elbashir SM,Harborth J,Lendeckel W,et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature,2001,411(6836):494-8.
[11] Elbashir SM,Harborth J,Weber K,et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs[J].Methods.2002,26(2):199-213.
[12] Harborth J,Elbashir SM,Bechert K,et al. Identification of essential genes in cultured mammalian cells using small interfering RNAs[J]. J Cell Sci,2001,114(24):4557-65.
[13] Timmons L,Court DL,Fire A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans[J]. Gene,2001,263(1-2):103-12.
[14] Paul CP,Good PD,Winer I,et al. Effective expression of small interfering RNA in human cells[J]. Nat Biotechnol,2002,20(5):505-8.
[15] Lee NS,Dohjima T,Bauer G,et al. Expression of small interfering RNAs targeted against HIV-l rev transcripts in human cells[J]. Nat Biotechnol,2002,20(5):500-5.
[16] Miyagishi M,Taira K. U6 promoter-driven siRNAs with four uridine 3＇ overhangs efficiently suppress targeted gene expression in mammalian cells[J]. Nat Biotechnol,2002,20(5):497-500.
[17] Mourrain P,Beclin C,Elmayan T,et al. Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance[J]. Cell,2000,101(5):533-42.
[18] Wu-Scharf D,Jeong B,Zhang C,et al. Transgenic and transpose silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase[J]. Science,2000,290(5494):1159-62.
[19] Catalanotto C,Azzalin G,Macino G,et al. Gene silencing in worms and fungi[J]. Nature,2000,404(6775):245-9.
[20] Catalanotto C,Azzalin G,Macino G,et al. Involvement of small RNAs and role of the qde genes in the gene silencing pathway in Neurospora[J]. Genes Dev,2002,16(7):790-5.
[21] 王吉兴,姚军.SV40LTAg基因永生化软骨细胞体外培养的生物学特性[J].第一军医大学学报,2003,23(12):1338-40.Wang JX,Yao J. Observation of the biological behavior of In vitrocultured immotalized chondrocytes induced by SV40LTAg gene transfection[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao,2003,23(12):1338-40.
[22] 欧阳平,陈慧萍,赖文岩,等.金钱鱼凋亡相关基因SaAR对NIH-3T3细胞凋亡的影响[J].第一军医大学学报,2003,23(11):1137-8.Ouyang P,ChenHP,Lai WY,et al.Effects ofScatophagus argus apoptosis-related gene on apoptosis ofNIH-3T3 cells[J]. J First Mil MedUniv/DiYi Jun Yi Da Xue Xue Bao,2003,23(11):1137-8.
[23] 罗勇,高建华,赵菲.Fas基因转染瘢痕疙瘩成纤维细胞并诱导其凋亡的实验研究[J].第一军医大学学报,2003,23(10):1015-7.Luo Y,Gao JH,Zhao F. Apoptosis of keloid-derived fibroblasts induced by Fas gene transfection[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao,2003,23(10):1015-7.