High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin
High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin
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DOI：
10.1006/dbio.1997.8779
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发表时间：
1997-12-15
影响因子：
2.7
通讯作者：
Eguchi, G
中科院分区：
文献类型：
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作者：
Higashijima, S;Okamoto, H;Eguchi, G
尽管有许多关于转基因斑马鱼的报道，但还没有关于转基因斑马鱼的报道，其中基因在斑马鱼来源的启动子的控制下。也没有关于转基因斑马鱼的报道，其中该基因是在组织特异性启动子/增强子的控制下。为了研究是否有可能产生在特定组织中可靠地表达报告基因的转基因斑马鱼，我们已经分离了斑马鱼肌肉特异性肌动蛋白（Lu-actin）启动子，并产生了转基因斑马鱼，其中绿色荧光蛋白（GFP）报告基因由该启动子驱动。总共产生了41个表达GFP的转基因株系，频率高达21%（41/194），并且GFP在几乎所有的株系（40/41）的整个肌肉细胞中特异性表达。不表达的转基因株系是罕见的。这表明，组织特异性启动子可以可靠地驱动报告基因在转基因斑马鱼中的表达的方式相同的基因的内源表达的控制。GFP的表达水平在品系之间变化很大;即，荧光在一些谱线中非常弱，而在另一些谱线中非常高。我们还分离了斑马鱼细胞骨架β-肌动蛋白启动子，并使用β-肌动蛋白-GFP构建体产生转基因斑马鱼。在产生的所有四个系中，GFP像β-肌动蛋白基因一样在整个身体中表达，表明在这种情况下也可以实现一致的表达。在本研究中，我们还研究了可能影响转基因频率或表达水平的因素的影响。获得了以下结果：（i）注射胚胎中GFP的表达水平与转基因频率没有强相关性;（ii）NLS肽的作用与转基因频率无关。（SV 40 T抗原核定位序列），其已被建议促进转基因转移到胚胎细胞核中，仍然是难以捉摸的;（iii）置于构建体上游的质粒载体序列可能降低报告基因的表达水平。(C)北京：科学出版社.
Despite a number of reports on transgenic zebrafish, there have been no reports on transgenic zebrafish in which the gene is under the control of a promoter of zebrafish origin. Neither have there been reports on transgenic zebrafish in which the gene is under the control of a tissue-specific promoter/enhancer. To investigate whether it is possible to generate transgenic zebrafish which reliably express a reporter gene in specific tissues, we have isolated a zebrafish muscle-specific actin (Lu-actin) promoter and generated transgenic zebrafish in which the green fluorescent protein (GFP) reporter gene was driven by this promoter. In total, 41 GFP-expressing transgenic lines were generated with a frequency of as high as 21% (41 of 194), and GFP was specifically expressed throughout muscle cells in virtually all of the lines (40 of 41). Nonexpressing transgenic lines were rare. This demonstrates that a tissue-specific promoter can reliably drive reporter gene expression in transgenic zebrafish in a manner identical to the control of the endogeneous expression of the gene. Levels of GFP expression varied greatly from line to line; i.e., fluorescence was very weak in some lines, while it was extremely high in others. We also isolated a zebrafish cytoskeletal beta-actin promoter and generated transgenic zebrafish using a beta-actin-GFP construct. In all of the four lines generated, GFP was expressed throughout the body like the beta-actin gene, demonstrating that consistent expression could also be achieved in this case. In the present study, we also examined the effects of factors which potentially affect the transgenic frequency or expression levels. The following results were obtained: (i) expression levels of GFP in the injected embryo were not strongly correlated to transgenic frequency; (ii) the effect of the NLS peptide (SV40 T antigen nuclear localization sequence), which has been suggested to facilitate the transfer of a transgene into embryonic nuclei, remained to be elusive; (iii) a plasmid vector sequence placed upstream of the construct might reduce the expression levels of the reporter gene. (C) 1997 Academic Press.