利用tol2介导的基因和增强子捕获方法对基因和基因组进行分析。
Analysis of genes and genome by the tol2-mediated gene and enhancer trap methods.
作者信息
Urasaki Akihiro, Kawakami Koichi
机构信息
Division of Molecular and Developmental Biology, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka, 411-8540, Japan.
出版信息
Methods Mol Biol. 2009;546:85-102. doi: 10.1007/978-1-60327-977-2_6.
The Tol2 transposon system can create insertions in the zebrafish genome efficiently. By using this system, the gene trap and enhancer trap methods have been developed. The gene trap and enhancer trap constructs contain the green fluorescent protein (GFP) reporter gene or the yeast Gal4 transcription activator gene. By creating random integrations of these constructs in the genome, transgenic fish expressing the GFP gene or the Gal4 gene in specific cells, tissues or organs are generated. These fish are valuable resources for developmental biology. Especially, the Gal4-expressing transgenic fish can be used to ectopically express any gene of interest placed downstream of the Gal4 recognition sequence, UAS, and thereby allow visualization, modification or ablation of the Gal4-expressing cells. In this chapter, we will describe how the gene trap and enhancer trap screens can be performed and how the transposon insertions created by these methods can be analyzed.
Tol2转座子系统能够高效地在斑马鱼基因组中产生插入。利用该系统,已开发出基因捕获和增强子捕获方法。基因捕获和增强子捕获构建体包含绿色荧光蛋白(GFP)报告基因或酵母Gal4转录激活基因。通过在基因组中随机整合这些构建体,可产生在特定细胞、组织或器官中表达GFP基因或Gal4基因的转基因鱼。这些鱼是发育生物学的宝贵资源。特别是,表达Gal4的转基因鱼可用于异位表达位于Gal4识别序列UAS下游的任何感兴趣基因,从而实现对表达Gal4的细胞进行可视化、修饰或消融。在本章中,我们将描述如何进行基因捕获和增强子捕获筛选,以及如何分析通过这些方法产生的转座子插入。