Arnone M I, Bogarad L D, Collazo A, Kirchhamer C V, Cameron R A, Rast J P, Gregorians A, Davidson E H
Division of Biology and Stowers Institute for Medical Research, California Institute of Technology, Pasadena 91125, USA.
Development. 1997 Nov;124(22):4649-59. doi: 10.1242/dev.124.22.4649.
The use of Green Fluorescent Protein (GFP) as a reporter for expression transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development. A GFP coding sequence was associated with three different previously studied cis-regulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic and secondary mesenchyme, and the Endo16 gene, expressed in vegetal plate, archenteron and midgut. We demonstrate that the sensitivity with which expression can be detected is equal to or greater than that of whole-mount in situ hybridization applied to detection of CAT mRNA synthesized under the control of the same cis-regulatory systems. However, in addition to the important feature that it can be visualized nondestructively in living embryos, GFP has other advantages. First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connections between cells, which in sea urchin embryos is particularly useful for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme. Second, GFP expression can be dramatically visualized in postembryonic larval tissues. This brings postembryonic larval developmental processes for the first time within the easy range of gene transfer analyses. Third, GFP permits identification and segregation of embryos in which the clonal incorporation of injected DNA has occurred in any particular desired region of the embryo. Thus, we show explicitly that, as expected, GFP transgenes are incorporated in the same nuclei together with other transgenes with which they are co-injected.
利用绿色荧光蛋白（GFP）作为报告基因来表达转基因，为海胆发育过程中基因调控的研究开辟了几种新的实验策略。一个GFP编码序列与三个先前研究过的不同顺式调控系统相关联，即分别在造骨间充质中表达的SM50基因、在原肠、造骨和次生间充质中表达的CyIIa基因，以及在植物极板、原肠和中肠中表达的Endo16基因。我们证明，检测到的表达灵敏度等于或高于应用于检测在相同顺式调控系统控制下合成的CAT mRNA的全胚胎原位杂交。然而，除了能够在活胚胎中无损可视化这一重要特征外，GFP还有其他优点。首先，它甚至能在精细的细胞质丝内自由扩散，从而揭示细胞间的连接，这在海胆胚胎中对于观察在合胞体造骨间充质中起作用的调控系统特别有用。其次，GFP表达能在胚胎后期幼虫组织中显著可视化。这首次将胚胎后期幼虫发育过程带入基因转移分析的容易范围。第三，GFP允许识别和分离那些在胚胎的任何特定期望区域发生了注射DNA克隆整合的胚胎。因此，我们明确表明，正如预期的那样，GFP转基因与共注射的其他转基因一起被整合到相同的细胞核中。
