Lentivector-mediated RNAi efficiently downregulates
expression of murine cdk4 gene

doi:10.1007/s11684-009-0050-5

Front. Med.

2009, Vol. 3

Issue (3) : 287-291 https://doi.org/10.1007/s11684-009-0050-5

Research articles

Lentivector-mediated RNAi efficiently downregulates expression of murine cdk4 gene

Feng JIANG PhD ¹, Xuezhen WANG PhD ², Zheng XUE MD ², Suming ZHANG PhD ², Siyu FANG BM ², Min ZHANG MD, PhD ²,

1.Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China；Department of Neurology, Inner Mongolia Hospital, Hohhot 010017, China; 2.Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;

Download: PDF(158 KB)
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

Abstract In order to explore the role of cyclin-dependent kinase 4 (cdk4) in neurodegenerative diseases, lentiviral-delivered RNA interference (RNAi) was used to silence the expression of the murine cdk4 gene in vitro. Three cdk4-shRNAs of mouse and a negative sequence were designed. After synthesis and annealing, double strand oligonucleotides were cloned into a linearized pSIH1-H1-copGFP shRNA vector. It was confirmed by polymerase chain reaction (PCR) and sequencing that three pairs of cdk4-shRNAs and a negative shRNA were correctly inserted into the pSIH1-H1-copGFP vector. The above recombinants were transfected by lipofectamine into BV-2 cells. The gene silencing efficacy rates of the 3 targets were compared by Western blotting. The cdk4-siRNA2 was the most effective in silencing cdk4. The optimized pSIH1-cdk4-siRNA2 and pSIH-negative-siRNA were co-transfected into 293T cells with the lentiviral packaging plasmids respectively. The culture supernatant was harvested and condensed at the 24th and 48thh after transfection. Interference efficiency of the lentivirus expressing cdk4-siRNA was determined by reverse transcriptase-PCR (RT-PCR) and Western blotting in BV-2 cells. Lentivector-mediated RNAi could efficiently down-regulate the expression of the murine cdk4 gene in vitro, which provides a potential tool for studying and treating cdk4-related diseases.

Keywords cyclin-dependent kinase 4 RNA interference plasmid lentiviral vector

Issue Date: 05 September 2009

Cite this article:

Feng JIANG PhD,Xuezhen WANG PhD,Zheng XUE MD, et al. Lentivector-mediated RNAi efficiently downregulates expression of murine cdk4 gene [J]. Front. Med., 2009, 3(3): 287-291.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0050-5
https://academic.hep.com.cn/fmd/EN/Y2009/V3/I3/287

	Buée L, Bussière T, Buée-Scherrer V, Delacourte A, Hof P R. Tau protein isoforms, phosphorylationand role in neurodegenerative disorders. Brain Res Brain Res Rev, 2000, 33(1): 95―130 doi: 10.1016/S0165-0173(00)00019-9
	Giovanni A, Wirtz-Brugger F, Keramaris E, Slack R, Park D S. Involvement of cell cycle elements, cyclin-dependentkinases, pRb, and E2F x DP, in B-amyloid-induced neuronal death. J Biol Chem, 1999, 274(27): 19011―19016 doi: 10.1074/jbc.274.27.19011
	Vincent I, Pae C I, Hallows J L. The cell cycle and human neurodegenerative disease. Prog Cell Cycle Res, 2003, 5: 31―41
	Smith P D, O'Hare M J, Park D S. Emerging pathogenic role for cyclin dependent kinasesin neurodegeneration. Cell Cycle, 2004, 3(3): 289―291
	Jorda E G, Verdaguer E, Canudas A M, Jiménez A, Bruna A, Caelles C, Bravo R, Escubedo E, Pubill D, Camarasa J, Pallàs M, Camins A. Neuroprotectiveaction of flavopiridol, a cyclin-dependent kinase inhibitor, in colchicine-inducedapoptosis. Neuropharmacology, 2003, 45(5): 672―683 doi: 10.1016/S0028-3908(03)00204-1
	Elbashir S M, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interferencein cultured mammalian cells. Nature, 2001, 411(6836): 494―498 doi: 10.1038/35078107
	Wilda M, Fuchs U, Wössmann W, Borkhardt A. Killingof leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene, 2002, 21(37): 5716―5724 doi: 10.1038/sj.onc.1205653
	Jacque J M, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference. Nature, 2002, 418(6896): 435―438 doi: 10.1038/nature00896
	Shlomai A, Shaul Y. Inhibition of hepatitis Bvirus expression replication by RNA interference. Hepatology, 2003, 37(4): 764―770 doi: 10.1053/jhep.2003.50146
	Cann A J. RNA viruses. A practical approach. Oxford: OxfordUniversity Press, 2000
	Albertin G, Ruggero M, Guidolin D, Nussdorfer G G. Gene silencing of human RAMP2 mediated by short-interfering RNA. Int J Mol Med, 2006, 18(4): 531―535
	Abbas-Terki T, Blanco-Bose W, Déglon N, Pralong W, Aebischer P. Lentiviral-mediated RNA interference. Hum Gene Ther, 2002, 13(18): 2197―2201 doi: 10.1089/104303402320987888
	Hommel J D, Sears R M, Georgescu D, Simmons D L, DiLeone R J. Local gene knockdown in thebrain using viral-mediated RNA interference. Nat Med, 2003, 9(12): 1539―1544 doi: 10.1038/nm964
	Stewart S A, Dykxhoorn D M, Palliser D, Mizuno H, Yu E Y, An D S, Sabatini D M, Chen I S, Hahn W C, Sharp P A, Weinberg R A, Novina C D. Lentivirus-delivered stable gene silencing by RNAi inprimary cells. RNA, 2003, 9(4): 493―501 doi: 10.1261/rna.2192803
	Ong W Y, Kumar U, Switzer R C, Sidhu A, Suresh G, Hu C Y, Patel S C. Neurodegeneration in Niemann-Picktype C disease mice. Exp Brain Res, 2001, 141(2): 218―231 doi: 10.1007/s002210100870
	Khatib Z A, Matsushime H, Valentine M, Shapiro D N, Sherr C J, Look A T. Coamplification of the CDK4 gene with MDM2 and GLI inhuman sarcomas. Cancer Res, 1993, 53(22): 5535―5541
	Bu B T, Wang X Z, Zhang Y H. Activation of cdk4/cyclin D1 pathway participates inthe neuronal degeneration of murine niemann-pick disease type C. Xi Bao Sheng Wu Xue Za Zhi, 2005, 27(4): 435―440 (in Chinese)
	McCaffrey A P, Nakai H, Pandey K, Huang Z, Salazar F H, Xu H, Wieland S F, Marion P L, Kay M A. Inhibitionof hepatitis B virus in mice by RNA interference. Nat Biotechnol, 2003, 21(6): 639―644 doi: 10.1038/nbt824
	Smith-Arica J R, Bartlett J S. Gene therapy: recombinantadeno-associated virus vectors. Curr CardiolRep, 2001, 3(1): 43―49 doi: 10.1007/s11886-001-0009-x
	Rubinson D A, Dillon C P, Kwiatkowski A V, Sievers C, Yang L, Kopinja J, Rooney D L, Zhang M, Ihrig M M, McManus M T, Gertler F B, Scott M L, Van Parijs L. Alentivirus-based system to functionally silence genes in primary mammaliancells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003, 33(3): 401―406 doi: 10.1038/ng1117
	Sinn P L, Sauter S L, McCray P B Jr. Gene therapy progress and prospects: development of improvedlentiviral and retroviral vectors- design, biosafety, and production. Gene Ther, 2005, 12(14): 1089―1098 doi: 10.1038/sj.gt.3302570
	Dull T, Zufferey R, Kelly M, Mandel R J, Nguyen M, Trono D, Naldini L. Athird-generation lentivirus vector with a conditional packaging system. J Virol, 1998, 72(11): 8463―8471
	Wiznerowicz M, Trono D. Conditional suppression ofcellular genes: lentivirus vector mediated drug-inducible RNA interference. J Virol, 2003, 77(16): 8957―8961 doi: 10.1128/JVI.77.16.8957-8951.2003
	Pfeifer A, Eigenbrod S, Al-Khadra S, Hofmann A, Mitteregger G, Moser M, Bertsch U, Kretzschmar H. Lentivector-mediated RNAiefficiently suppresses prion protein and prolongs survival of scrapie-infectedmice. J Clin Invest, 2006, 116(12): 3204―3210 doi: 10.1172/JCI29236
	Yang Z, Cloud A, Hughes D, Johnson L F. Stable inhibition of human thymidylate synthase expression followingretroviral introduction of an siRNA gene. Cancer Gene Ther, 2006, 13(1): 107―114 doi: 10.1038/sj.cgt.7700880

[1]	Hui XIAO PhD, Ming TIAN MM, Junna GE MM, Xin Wei MD, Zhaoming LI MM, Xiaolan LI MS, Deding TAO PhD, Junbo HU MD, Jianping GONG MD, . The role of CDK1 siRNA interference in cell cycle and cell apoptosis[J]. Front. Med., 2009, 3(4): 384-389.
[2]	Cuihong ZHENG, Yunxia WU, Guangying HUANG, Wei WANG. Gene silencing efficiency of shRNA expression vectors targeting Cx43 in vitro[J]. Front Med Chin, 2009, 3(2): 130-135.
[3]	Zhengjuan LIU, Jie BIAN, Yuchuan WANG, Yongli ZHAO, Dong YAN, Xiaoxia WANG. Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene[J]. Front Med Chin, 2009, 3(1): 57-60.
[4]	ZHANG Shilong, ZENG Fuqing, PENG Shibo, WANG Liang. Effect on proliferation and apoptosis of T24 cell lines via silencing DNMT1 with RNA interference[J]. Front. Med., 2008, 2(4): 374-379.
[5]	LIU Hongchun, CAO Zhongwei, JIN Jianjun, WANG Jiyao. Blockage of receptor-interacting protein 2 expression by small interfering RNA in murine macrophages[J]. Front. Med., 2008, 2(2): 166-170.
[6]	ZHANG Jingqun, SUN Honglei, MA Yexin, WANG Daowen. Effects of RNA interference targeting angiotensin 1a receptor on blood pressure and cardiac hypertrophy of rats with renovascular hypertension[J]. Front. Med., 2008, 2(1): 19-24.
[7]	JI Yewei, NIE Bin, LI Ping, ZHOU Yuanguo, XU Xiaoyu. Construction of Hsp90β gene specific silencing plasmid and its transfection efficiency[J]. Front. Med., 2007, 1(3): 253-257.

Viewed

Full text

Abstract

Cited

Shared

Discussed