小鼠<em>FABP4</em>基因启动子驱动红色荧光蛋白载体的构建及在牛体细胞中的表达

doi:10.7668/hbnxb.2016.06.005

摘要/Abstract

摘要： 小鼠脂肪型脂肪酸结合蛋白（FABP4）的启动子/增强子元件是脂肪组织特异性启动元件，为了鉴定该元件在牛体细胞中的启动效果，首先以小鼠肝脏DNA为模板，通过PCR克隆得到5.9 kb的FABP4基因启动子片段，连入pMD19-T载体后进行酶切及测序鉴定，经EcoT 22I酶切去除非核心启动子区后精简为2.3 kb的片段，通过Sac Ⅱ酶切将5.9 kb片段和2.3 kb片段的启动子连入红色荧光蛋白载体pDs-Red 2-1的多克隆位点，构建为表达质粒pMF5.9-Red和pMF2.3-Red，利用脂质体法将上述载体分别转染牛脂肪间充质干细胞及牛胎儿成纤维细胞，24 h后实时定量PCR检测红色荧光蛋白转录水平。结果表明，克隆得到的5.9 kb小鼠FABP4启动子片段酶切及测序结果正确，与红色荧光蛋白载体相连的载体pMF5.9-Red和pMF2.3-Red酶切结果与预期相符，表达载体构建成功，实时定量PCR结果显示以上2种细胞在转染后24 h红色荧光蛋白均有表达，且pMF2.3-Red的转录水平是pMF5.9-Red的2倍以上。成功构建了小鼠FABP4启动子驱动红色荧光蛋白表达载体pMF5.9-Red和pMF2.3-Red，5.9 kb片段和2.3 kb片段均可驱动外源基因在牛体细胞中转录，且2.3 kb片段启动效率高于5.9 kb片段。

关键词: 小鼠FABP4启动子, 载体构建, 牛体细胞

Abstract: The promoter/enhancer cassette of mouse adipocyte-type fatty acid binding protein(FABP4) was widely used as the adipocyte specific element.To detect the effect of mFABP4 promoter on gene expression in bovine cells,5.9 kb mFABP4 promoter fragment was cloned from mouse liver genome and identified by digestion and sequencing after ligated to pMD19-T vector.By digestion of enzyme EcoT 22I the non-core promoter fragment was deleted from the 5.9 kb fragment and another 2.3 kb core promoter fragment was obtained.The 5.9 kb and 2.3 kb fragment were inserted into the multiple cloning site of plasmid pDs-Red 2-1 by enzyme Sac Ⅱ,respectively.As a result the vectors,pMF5.9-Red and pMF2.3-Red,were constructed successfully.By lipofection,the constructed pMF5.9-Red and pMF2.3-Red vectors were transfected into bovine adipose-derived stem cells and bovine embryo fibroblast cells,respectively.24 h after transfection,Real-time PCR was applied to detect the transcription of red fluorescent protein in transfected cells.Results showed that the sequence of cloned 5.9 kb fragment was correct,and the 5.9 kb and the 2.3 kb fragments were correctly ligated into vectors.The mRNA of red fluorescent protein was both detected in transfected bovine adipose-derived stem cells and bovine embryo fibroblast cells,and the expression level displayed 2 fold higher in pMF2.3-Red than that of pMF5.9-Red.In conclusion,this study constructed the expression vector pMF5.9-Red and pMF2.3-Red with 5.9 kb or 2.3 kb fragment of mouse FABP 4 gene as promoter region could initiate the transcription of foreign genes in bovine cells and the transcription efficiency by 2.3 kb promoter fragment was higher than that of 5.9 kb promoter fragment.

Key words: Mouse FABP4 promoter, Vector construction, Cattle cells

中图分类号:

Q782

岳永莉, 于海泉. 小鼠FABP4基因启动子驱动红色荧光蛋白载体的构建及在牛体细胞中的表达[J]. 华北农学报, 2016, 31(6): 26-30. doi: 10.7668/hbnxb.2016.06.005.

YUE Yongli, YU Haiquan. Construction of Red Fluorescent Protein Driven by Mouse FABP4 Promoter and Expression in Bovine Cells[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(6): 26-30. doi: 10.7668/hbnxb.2016.06.005.

http://www.hbnxb.net/CN/Y2016/V31/I6/26

参考文献

[1] Bertolini L R,Meade H,Lazzarotto C R,et al.The transgenic animal platform for biopharmaceutical production[J].Transgenic Res,2016,25(3):329-343.
[2] Barlow C,Schroeder M,Lekstrom-Himes J,et al.Targeted expression of Cre recombinase to adipose tissue of transgenic mice directs adipose-specific excision of loxP-flanked gene segments[J].Nucleic Acids Res,1997,25(12):2543-2545.
[3] Abel E D,Peroni O,Kim J K,et al.Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver[J].Nature,2001,409(6821):729-733.
[4] He W,Barak Y,Hevener A,et al.Adipose-specific peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle[J].Proc Natl Acad Sci U S A,2003,100(26):15712-15717.
[5] Imai T,Jiang M,Chambon P,et al.Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes[J].Proc Natl AcadSci U S A,2001,98(1):224-228.
[6] Zhang X,Xu A,Chung S K,et al.Selective inactivation of c-Jun NH2-terminal kinase in adipose tissue protects against diet-induced obesity and improves insulin sensitivity in both liver and skeletal muscle in mice[J].Diabetes,2011,60(2):486-495.
[7] Wong K E,Kong J,Zhang W,et al.Targeted expression of human vitamin D receptor in adipocytes decreases energy expenditure and induces obesity in mice[J].J Biol Chem,2011,286(39):33804-33810.
[8] Kanda H,Tateya S,Tamori Y,et al.MCP-1 contributes to macrophage infiltration into adipose tissue,insulin resistance,and hepatic steatosis in obesity[J].J Clin Invest,2006,116(6):1494-1505.
[9] Storch J,Thumser A E.Tissue-specific functions in the fatty acid-binding protein family[J].J Biol Chem,2010,285(43):32679-32683.
[10] Shin J,Li B,Davis M E,et al.Comparative analysis of fatty acid-binding protein 4 promoters:conservation of peroxisome proliferator-activated receptor binding sites[J].J Anim Sci,2009,87(12):3923-3934.
[11] Ross S R,Graves R A,Greenstein A,et al.A fat-specific enhancer is the primary determinant of gene expression for adipocyte P2in vivo[J].Proc Natl Acad Sci U S A,1990,87(24):9590-9594.
[12] Rival Y,Stennevin A,Puech L,et al.Human adipocyte fatty acid-binding protein (aP2) gene promoter-driven reporter assay discriminates nonlipogenic peroxisome proliferator-activated receptor gamma ligands[J].J Pharmacol Exp Ther,2004,311(2):467-475.
[13] Jung E M,An B S,Kim Y K,et al.Generation of porcine fibroblasts overexpressing 11beta-HSD1 with adipose tissue-specific aP2 promoter as a porcine model of metabolic syndrome[J].Mol Med Rep,2013,8(3):751-756.
[14] Jurczak M J,Danos A M,Rehrmann V R,et al.Transgenic overexpression of protein targeting to glycogen markedly increases adipocytic glycogen storage in mice[J].Am J Physiol Endocrinol Metab,2007,292(3):952-963.
[15] Shi S Y,Luk C T,Brunt J J,et al.Adipocyte-specific deficiency of Janus kinase (JAK) 2 in mice impairs lipolysis and increases body weight,and leads to insulin resistance with ageing[J].Diabetologia,2014,57(5):1016-1026.
[16] Wang Z V,Deng Y,Wang Q A,et al.Identification and characterization of a promoter cassette conferring adipocyte-specific gene expression[J].Endocrinology,2010,151(6):2933-2939.
[17] Devine J H,Eubank D W,Clouthier D E,et al.Adipose expression of the phosphoenolpyruvate carboxykinase promoter requires peroxisome proliferator-activated receptor gamma and 9-cis-retinoic acid receptor binding to an adipocyte-specific enhancer in vivo[J].J Biol Chem,1999,274(19):13604-13612.
[18] Kershaw E E,Morton N M,Dhillon H,et al.Adipocyte-specific glucocorticoid inactivation protects against diet-induced obesity[J].Diabetes,2005,54(4):1023-1031.
[19] Pravenec M,Kazdova L,Landa V,et al.Transgenic and recombinant resistin impair skeletal muscle glucose metabolism in the spontaneously hypertensive rat[J].J Biol Chem,2003,278(46):45209-45215.
[20] 陈建文,刘星,桂涛,等. fat-1 基因脂肪组织特异性表达载体的构建及其山羊转基因细胞系的建立[J].安徽农业大学学报,2012,39(5):746-750.
[21] Vargas D,Shimokawa N,Kaneko R,et al.Regulation of human subcutaneous adipocyte differentiation by EID1[J].J Mol Endocrinol,2016,56(2):113-122.

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小鼠FABP4基因启动子驱动红色荧光蛋白载体的构建及在牛体细胞中的表达

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小鼠FABP4基因启动子驱动红色荧光蛋白载体的构建及在牛体细胞中的表达

Construction of Red Fluorescent Protein Driven by Mouse FABP4 Promoter and Expression in Bovine Cells

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