Odell J T, Hoopes J L, Vermerris W
Agricultural Products, DuPont, Experimental Station, Wilmington, Delaware 19880-0402.
Plant Physiol. 1994 Oct;106(2):447-58. doi: 10.1104/pp.106.2.447.
The Cre/lox site-specific recombination system was used to activate a transgene in a tissue-specific manner. Cre-mediated activation of a beta-glucuronidase marker gene, by removal of a lox-bounded blocking fragment, allowed the visualization of the activation process. By using seed-specific promoters, the timing and efficiency of gene activation could be followed within the developing tobacco (Nicotiana tabacum) embryo. To serve as a basis for analyzing gene expression after-Cre-mediated activation, the timing and patterns of expression of the promoters of the genes encoding French bean (Phaseolus vulgaris) beta-phaseolin and the alpha' subunit of soybean (Glycine max) beta-conglycinin, as well as the cauliflower mosaic virus 35S promoter, were studied in developing transgenic tobacco embryos using the same visual marker. These seed-specific promoters were expressed earlier than anticipated. The 35S promoter was expressed earlier than the seed-specific promoters, but not in globular-stage embryos. Cre-mediated gene activation occurred approximately 1 d after promoter activation, based on developmental staging, and spread progressively throughout the embryo. The timing of gene activation was varied by altering Cre expression. Efficient Cre expression ultimately directed gene activation throughout the model tissue, whereas inefficient Cre expression resulted in mosaic tissue. Limited gene activation provides a system for cell lineage and developmental analyses.
Cre/lox位点特异性重组系统被用于以组织特异性方式激活转基因。通过去除lox边界的阻断片段，Cre介导的β-葡萄糖醛酸酶标记基因激活使得激活过程可视化。利用种子特异性启动子，可以在发育中的烟草（Nicotiana tabacum）胚胎中追踪基因激活的时间和效率。为了作为分析Cre介导的激活后基因表达的基础，使用相同的视觉标记，在发育中的转基因烟草胚胎中研究了编码菜豆（Phaseolus vulgaris）β-菜豆蛋白和大豆（Glycine max）β-伴大豆球蛋白α'亚基的基因启动子以及花椰菜花叶病毒35S启动子的表达时间和模式。这些种子特异性启动子的表达比预期更早。35S启动子比种子特异性启动子表达更早，但在球形期胚胎中不表达。基于发育阶段，Cre介导的基因激活在启动子激活后约1天发生，并逐渐扩散到整个胚胎。通过改变Cre表达来改变基因激活的时间。高效的Cre表达最终导致整个模型组织中的基因激活，而低效的Cre表达导致镶嵌组织。有限的基因激活为细胞谱系和发育分析提供了一个系统。
