本课题组前期研究中, 利用斑马鱼cmlc2 (Cardiac myosin light chain 2)基因启动子构建了一个用于斑马鱼心脏组织特异表达外源基因的转基因表达载体pTol2-cmlc2-IRES-EGFP。文章利用该载体构建了一个稳定表达EGFP的转基因斑马鱼品系, 并初步分析了EGFP的表达对该转基因斑马鱼品系的心脏发育和功能的影响。结果表明, 在建立的转基因斑马鱼品系早期胚胎发育过程中, 绿色荧光信号在心脏中特异表达, 该表达模式与原位杂交分析的cmlc2的表达模式结果相同; 该转基因斑马鱼品系的心脏形态及发育生长正常; 进一步通过M-Mode分析心脏生理学功能的结果表明：该转基因品系心动周期、心率、收缩与舒张表面积及表面积缩短率等重要生理指标与正常野生型的斑马鱼对照组相比没有显著差别。以上结果表明该转基因品系中绿色荧光蛋白的表达对斑马鱼心脏的发育和功能没有影响。研究结果为进一步利用该载体建立外源目的基因转基因表达模型, 研究心脏表达基因的功能奠定了重要基础。
Using the promoter for cardiac myosin light chain 2 (cmlc2) gene, an expression vector pTol2-cmlc2-IRES-EGFP for making heart-specific expression of exogenous gene in transgenic zebrafish was generated previously. Here, we reported the construction of a transgenic zebrafish line which stably expresses EGFP using this vector, and the effects of EGFP on the heart development and cardiac function of this transgenic zebrafish line were preliminarily analyzed. The results showed that the green fluorescence signal of cmlc2:EGFP line under fluo-rescence microscopy specifically expressed in heart and faithfully recapitulated both the spatial and temporal expression patterns of endogenous cmlc2 gene revealed by in situ hybridization in the early developmental stages. The cardiac morphology and development of this transgenic zebrafish line remained to be normal. Furthermore, the heart mor-phology and physiological function of this transgenic line have been analyzed using M-mode analysis. The results showed that there was no significant difference between the cmlc2:EGFP and the wild type lines with respect to heart period, heart rate, diastolic surface area and systolic surface area, and fractional area change. No tachyarrhythmia was observed in the embryos from either line. Thus, the excessive expression of EGFP in this transgenic line seemed to exert no detrimental effects on the function and development of zebrfish hearts during early stages. Our study laid a foundation for the construc-tion of exogenous gene transgenic line using pTol2-cmlc2-IRES-EGFP vector to study the function of genes that expressed in heart.
[1] Shimada E, Kinoshita M, Murata K. Expression of cardiac myosin light chain 2 during embryonic heart development in medaka fish, Oryzias latipes, and phylogenetic relationship with other myosin light chains. Dev Growth Differ, 2009, 51(1): 1-16.
[2] Huang CJ, Tu CT, Hsiao CD, Hsieh FJ, Tsai HJ. Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev Dyn, 2003, 228(1): 30-40.
[3] Burns CG, Milan DJ, Grande EJ, Rottbauer W, MacRae CA, Fishman MC. High-throughput assay for small mole-cules that modulate zebrafish embryonic heart rate. Nat Chem Biol, 2005, 1(5): 263-264.
[4] Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 1996, 173(1): 33-38.
[5] Yang TT, Cheng LZ, Kain SR. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res, 1996, 24(22): 4592-4593.
[6] Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell, 2004, 7(1): 133-144.
[7] Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS, Parant JM, Yost HJ, Kanki JP, Chien CB. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn, 2007, 236(11): 3088-3099.
[8] 陈婷芳, 罗娜, 谢华平, 吴秀山, 邓云. 利用Tol2转座子构建斑马鱼心脏组织特异表达转基因载体及其表达分析. 生物工程学报, 2010, 26(2): 230-236.
[9] Martínez-Salas E. Internal ribosome entry site biology and its use in expression vectors. Curr Opin Biotech-nol, 1999, 10(5): 458-464.
[10] Wester?eld M. The Zebra?sh Book. Eugene: University of Oregon Press, 1995.
[11] Jowett T. Double in situ hybridization techniques in zebra?sh. Methods, 2001, 23(4): 345-358.
[12] Ocorr K, Perrin L, Lim HY, Qian L, Wu XS, Bodmer R. Genetic control of heart function and aging in Drosophila. Trends Cardiovasc Med, 2007, 17(5): 177-182.
[13] Ocorr K, Fink M, Cammarato A, Bernstein SI, Bodmer R. Semi-automated optical heartbeat analysis of small hearts. J Vis Exp, 2009, 16(31): 1435.
[14] Fink M, Callol-Massot C, Chu A, Ruiz-Lozano P, Bel-monte JC, Giles W, Bodmer R, Ocorr K. A new method for detection and quanti?cation of heartbeat parameters in Drosophila, zebrafish, and embryonic mouse hearts. Biotechniques, 2009, 46(2): 101-113.
[15] Wang YQ, Zhou JN, Ye XL, Wan YQ, Li YQ, Mo XY, Yuan WZ, Yan Y, Luo N, Wang ZQ, Fan XW, Deng Y, Wu XS. ZNF424, a novel human KRAB/C2H2 zinc finger protein, suppresses NFAT and p21 pathway. BMB Rep, 2010, 43(3): 212-218.
[16] 孟安明, 林硕. 利用GATA-2调控成分制备组织特异性表达GFP的转基因斑马鱼. 科学通报, 1999, 44(21): 2283-2287.
[17] Soboleski MR, Oaks J, Phalford W. Green fluorescent protein is a quantitative reporter of gene expression in in-dividual eukaryotic cells. FASEB J, 2005, 19(3): 440-442.
[18] Huang WY, Aramburu J, Douglas PS, Izumo S. Transgenic expression of green ?uorescence protein can cause dilated cardiomyopathy. Nat Med, 2000, 6(5): 482-483.
[19] Ho YL, Lin YH, Tsai IJ, Hsieh FJ, Tsai HJ. In vivo as-sessment of cardiac morphology and function in heart-speci?c green ?uorescent zebra?sh. J Formos Med Assoc, 2007, 106(3): 181-186.
[20] Huang W, Y Deng, Dong W, Yuan WZ, Wan YQ, Mo XY, Li YQ, Wang ZQ, Wang YQ, Ocorr K, Zhang B, Lin S, Wu XS. The effect of excess expression of GFP in a novel heart-specific green fluorescence in zebrafish regulated by nppa enhancer at early embryonic development. Mol Biol Rep, 2011, 38(2): 793-799.
