Pol Ⅱ型启动子K14实现组织特异RNAi

doi:10.3724/SP.J.1005.2011.00757

[an error occurred while processing this directive]

HEREDITAS ›› 2011, Vol. 33 ›› Issue (7): 757-762.doi: 10.3724/SP.J.1005.2011.00757

• en • Previous Articles Next Articles

shRNAs driven by K14 promoter induce tissue-specific RNA interference

DAI Rong^{1, 2}, SHEN Si-Jun¹, WAN Peng-Cheng^1,2, SHI Guo-Qing^1,2, MENG Qing-Yong³, LIU Shou-Ren^1,2

1. College of Animal Science, Shihezi University, Shihezi 832000, China 2. Breed & Biotechnology Key Laboratory of Sheep in Production and Construction Corps of Xinjiang, Xinjiang Agricultural Reclama-tion Academy, Shihezi 832000, China 3. State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100193, China

Received:2010-10-09 Revised:2011-01-26 Online:2011-07-20 Published:2011-07-25
Contact: LIU Shou-Ren E-mail:nkyysb@163.com

Abstract

Abstract: RNA interference is an efficient method for exploring gene function. Accumulating evidence suggests that RNA Pol II promoters can direct cell- or tissue-specific gene silencing. A eGFP-shRNA fusion construct transcribed from an RNA Pol II promoter (K14 promoter) was used to induce gene-specific shRNA silencing of BMP4 gene expression. Re-combinant vectors (pEGFP-C1-shRNA, psiCHECK-BMP4, and pEGFP-K14-shRNA) were constructed. Vectors pEGFP-C1-shRNA and psiCHECK-BMP4 were cotransfected into Hela cells (in vitro) and shRNA-induced inhibi-tion efficiency was tested by a luciferase assay. The results showed that all the six interference sequences inhibited the ex-pression of BMP4 with high efficiency (>60%), and the interference sequence 5# showed the highest efficiency. For in vivo screening of JB6-C41 cells transfected with vector pEGFP-K14-shRNA, the inhibition efficiency was assayed by quantitative RT-PCR and Western blotting analyses. The results showed that the mRNA and protein products of the exoge-nous BMP4 gene were efficiently and specifically inhibited. The efficiency of gene silencing was greater than 60%, except for sequence 3#. The declines in mRNA and protein expression levels were significantly correlated during gene si-lence by the shRNA. This system may be adapted for in vivo shRNA expression and gene silencing. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in the analysis of the mechanisms of hair follicle development in sheep.

Key words: tissue-specific, RNAi, K14 promoter, fusion construct, transgenic livestock

DAI Rong, CHEN Sai-Jun, MO Feng-Cheng, DAN Guo-Qiang, MENG Qiang-Yong, LIU Shou-Ren. shRNAs driven by K14 promoter induce tissue-specific RNA interference[J]. HEREDITAS, 2011, 33(7): 757-762.

References

[1] Kulessa H, Turk G, Hogan BLM. Inhibition of Bmp signaling affects growth and differentiation in the anagen hair follicle. EMBO J, 2000, 19(24): 6664-6674.
[2] Sharov AA, Weiner L, Sharova TY, Siebenhaar F, Atoyan R, Reginato AM, McNamara CA, Funa K, Gilchrest B A, Bris-sette JL, Botchkarev VA. Noggin overexpression inhibits eye-lid opening by altering epidermal apoptosis and differentiation. EMBO J, 2003, 22(12): 2992-3003.
[3] Guba U, Mecklenburg L, Cowin P, Kan L, O'Guin WM, D'Vizio D, Pestell RG, Paus R, Kessler JA. Bone morphogenetic protein signaling regulates postnatal hair follicle differentiation and cycling. Am J Pathol, 2004, 165(3): 729-740.
[4] Botchkarev AV, Botchkareva NV, Sharov AA, Funa K, Huber O, Gilchrest BA. Modulation of BMP signaling by noggin is required for induction of the secondary (nontylotrich) hair follicle. J Invest Dermatol, 2002, 118(1): 3-10.
[5] Andl T, Ahn K, Kairo A, Chu EY, Wine-Lee L, Reddy ST, Croft NJ, Cebra-Thomas JA, Metzger D, Chambon P, Lyons KM, Mishina Y, Seykora JT, Crenshaw EB, Millar SE. Epithe-lial Bmpr1a regulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development. Development, 2004, 131(10): 2257-2268.
[6] 张定校, 樊斌, 刘榜, 李奎. RNA干涉 (RNAi) 技术应用于哺乳动物细胞的研究策略. 遗传, 2005, 27(5): 839-844.
[7] Ventura A, Meissner A, Dillon CP, McManus M, Sharp PA, Van Parijs L, Jaenisch R, Jacks T. Cre-lox-regulated condi-tional RNA interference from transgenes. Proc Natl Acad Sci USA, 2004, 101(28): 10380-10385.
[8] Ling X, Li FZ. Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology. Biotechniques, 2004, 36(3): 450-460.
[9] 袁晶. 鸡细胞中基于载体的RNA干涉研究
[学位论文]. 北京: 中国农业大学, 2007.
[10] Yuan J, Wang XB, Zhang Y, Hu XX, Deng XM, Fei J, Li N. shRNA transcribed by RNA Pol II promoter induce RNA interference in mammalian cell. Mol Biol Rep, 2006, 33(1): 43-49.
[11] Hanukoglu I, Fuchs E. The cDNA sequence of a human epidermal keratin: Divergence of sequence but conservation of structure among intermediate filament proteins. Cell, 1982, 31(1): 243-252.
[12] Hanukoglu I, Fuchs E. The cDNA sequence of a type II cytoskeletal keratin reveals constant and variable structural domains among keratins. Cell, 1983, 33(3): 915-924.
[13] Vassar R, Rosenberg M, Ross S, Tyner A, Fuchs E. Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice. Proc Natl Acad Sci USA, 1989, 86(5): 1563-1567.
[14] Staggers WR, Paterson AJ, Kudlow JE. Sequence of the func-tional human keratin K14 promoter. Gene, 1995, 153(2): 297-298.
[15] Vassar R, Fuchs E. Transgenic mice provide new insights into the role of TGF-alpha during epidermal development and differentiation. Genes Dev, 1991, 5(5): 714-727.
[16] Turksen K, Kupper T, Degenstein L, Williams I, Fuchs E. In-terleukin 6: insights to its function in skin by overexpression in transgenic mice. Proc Natl Acad Sci USA, 1992, 89(11): 5068-5072.
[17] Williams IR, Kupper TS. Epidermal expression of intercellular adhesion molecule 1 is not a primary inducer of cutaneous in-flammation in transgenic mice. Proc Natl Acad Sci USA, 1994, 91(21): 9710-9714.
[18] 任红艳, 张兴举, 杨述林, 崔文涛, 唐中林, 李奎. c-Myc转基因小鼠构建的改良方法. 农业生物技术学报, 2008, 16(1): 37-40.
[19] 潘巍巍, 赖国旗, 宋方洲. 角蛋白启动子在人乳头瘤病毒的转基因小鼠中的研究进展. 四川动物, 2006, 25(4): 903-905.
[20] 王勇, 王峰超, 魏泓, 倪勇, 吴军, 高翔. 皮肤组织特异性表达hCTLA4-Ig转基因小鼠品系的建立. 遗传学报, 2005, 32(9): 916-922.

Metrics

Comments

www.chinagene.cn
备案号：京ICP备09063187号

shRNAs driven by K14 promoter induce tissue-specific RNA interference

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	Yuting Han, Bowen Xu, Yutong Li, Xinyi Lu, Xizhi Dong, Yuhao Qiu, Qinyun Che, Ruibao Zhu, Li Zheng, Xiaochen Li, Xu Si, Jianquan Ni. The cutting edge of gene regulation approaches in model organism Drosophila [J]. Hereditas(Beijing), 2022, 44(1): 3-14.
[2]	Yakun Song,Min Zhang,Qiaochu Wang,Yuli Peng,Fangxing Jia,Chunhong Yu. Laboratory design and practice for undergraduates: Using RNAi to modulate gene expression [J]. Hereditas(Beijing), 2019, 41(7): 653-661.
[3]	GONG Wen-Fang LU Li-Jing LIU Xin CHEN Xi-Wen CHEN De-Fu. Effect of silencing lycB gene on the carotenoid synthesis in Haematococcus pluvialis [J]. HEREDITAS, 2013, 35(2): 233-240.
[4]	ZHENG Wei-Hao, LIN Zhi-Qiang, ZHUO Min, DU Hong-Li, WANG Xiao-Ning. Research progress on influenza antiviral small RNAs [J]. HEREDITAS, 2012, 34(5): 526-532.
[5]	WANG Li, NA Wei, WANG Yu-Xiang, WANG Yan-Bo, WANG Ning, WANG Qi-Gui, LI Yu-Mao, LI Hui. Characterization of chicken PPARγ expression and its impact on adipocyte proliferation and differentiation [J]. HEREDITAS, 2012, 34(4): 454-464.
[6]	MA Sheng-Yun, BAI Yu, HAN Ning, WANG Jun-Hui, WENG Xiao-Yan, BIAN Hong-Wu, ZHU Mu-Yuan. Recent research progress of biogenesis and functions of miRNA [J]. HEREDITAS, 2012, 34(4): 383-388.
[7]	LIU Wu-Wen, GU Bin, DAN Guo-Qiang, LIN Jian-Gong, LIU Ka, MA Run-Lin. Cloning, expression analysis and RNA interference of FGF5 gene in sheep [J]. HEREDITAS, 2011, 33(9): 982-988.
[8]	HE Miao, WANG Zi-Wei. Current status and development of miRNA and siRNA research on gastric cancer [J]. HEREDITAS, 2011, 33(9): 925-930.
[9]	JIN Li-Hua, QI Zhuo. Drosophila spen protein: generation of ployclonal antibodies, functional analysis and tissue-specific expression [J]. HEREDITAS, 2011, 33(11): 1239-1244.
[10]	SHI Ming-Lei, ZHAO Zhi-Hu, WANG Yang, CHEN Hui-Peng. In vivo delivery of siRNA [J]. HEREDITAS, 2009, 31(7): 683-689.
[11]	LI Ruo, TIAN Yan-Yan, GAO Yong, WANG Shan, NIE Huan, LI Yu. Silencing LRP1B with huge transcript by RNAi method [J]. HEREDITAS, 2009, 31(1): 36-42.
[12]	LI Fu-Bing, DU Xiao-Lan, YU Ying, ZHAO Ling, HE Qi-Fen, CHEN Lin . Generation of bone morphogenetic protein 4 conditional RNA inter-ference mice [J]. HEREDITAS, 2008, 30(3): 341-346.
[13]	YAN Pei-Qiang, BAI Xian-Quan, WAN Xiu-Qing, GUO Zhao-Kui, LI Li-Jie, GONG Hai-Ying, CHU Cheng-Cai. Expression of TMV coat protein gene RNAi in transgenic tobacco plants confer immunity to tobacco mosaic virus infection [J]. HEREDITAS, 2007, 29(8): 1018-1018―1022.
[14]	TU Zhi-Ming, CHEN Lin, YANG Guang-Xiao, HE Guang-Yuan . Application of enzyme-labeled probe in testing of transgenic plant [J]. HEREDITAS, 2007, 29(12): 1533-1537.
[15]	YIN Xiu-Shan, ZHANG Ling-Qiang, HE Fu-Chu. The Application of RNAi Technology in Transgenic Mice [J]. HEREDITAS, 2006, 28(3): 351-356.