THE AEQUOREA-VICTORIA GREEN FLUORESCENT PROTEIN CAN BE USED AS A REPORTER IN LIVE ZEBRAFISH EMBRYOS
THE AEQUOREA-VICTORIA GREEN FLUORESCENT PROTEIN CAN BE USED AS A REPORTER IN LIVE ZEBRAFISH EMBRYOS
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DOI：
10.1006/dbio.1995.1265
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发表时间：
1995-09-01
影响因子：
2.7
通讯作者：
HOPKINS, N
中科院分区：
文献类型：
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作者：
AMSTERDAM, A;LIN, S;HOPKINS, N
来自刺胞动物维多利亚水母的绿色荧光蛋白（GFP）能够在没有外源添加底物的情况下产生荧光。在这里，我们证明了由非洲爪蟾延伸因子1 α增强子启动子驱动的GFP的cDNA可以在活斑马鱼胚胎上赋予荧光，无论是作为注射的质粒还是作为通过种系后的转基因。当注射到处于单细胞阶段的斑马鱼胚胎中时，该构建体在28 ℃下发育约4小时后开始表达可检测的GEP，在注射后至少3周，在胚胎中的许多组织类型的细胞中可以观察到中囊胚转变后约1小时的荧光。我们使用了三种不同的表达构建体，每种构建体都使用了一个修饰的ef 1 α增强子启动子，以产生12个转基因株系。12个品系中的8个，包括5个衍生自具有内含子的一个构建体的品系中的5个，在F1中表达可检测的荧光，并且在F2代中表达可检测的荧光。大多数表达系表现出非常相似的表达模式。通常，在受精后20小时之前，在转基因胚胎中看不到荧光，此时荧光在整个胚胎中均匀出现。荧光在24-36小时之间最明显，此后变得不太明显，除了在许多线中，强荧光在眼睛中保持可见至少5天。转基因的单个遗传拷贝足以在半合子F1和F2胚胎中产生可检测的荧光。(C)出版社：Academic Press
The green fluorescent protein (GFP) from the cnidarian Aequorea victoria is capable of producing fluorescence without an exogenously added substrate. Here we demonstrate that a cDNA for GFP driven by a Xenopus elongation factor 1 alpha enhancer-promoter can confer fluorescence upon live zebrafish embryos, either as an injected plasmid or as a transgene after passage through the germline. When injected into zebrafish embryos at the one-cell stage, this construct starts to express detectable GEP after about 4 hr of development at 28 degrees C, about 1 hr after the midblastula transition fluorescence can be observed in cells of many tissue types in the embryo for at least 3 weeks after injection. We used three different expression constructs, each employing a modified ef1 alpha enhancer-promoter, to generate 12 transgenic lines. Eight of the 12 lines, including 5 of 5 derived from one construct with an intron, express detectable fluorescence in the F1 and, where tested, in the F2 generation. Most expressing lines showed very similar expression patterns. Generally, fluorescence is not seen in the transgenic embryos before 20 hr postfertilization, at which point it appears uniformly throughout the embryo. fluorescence is most visible between 24-36 hr, and it becomes less visible after this, except that in many lines strong fluorescence remains visible in the eye for at least 5 days. A single inherited copy of the transgene is sufficient to produce detectable fluorescence in hemizygous F1 and F2 embryos. (C) 1995 Academic Press, Inc.