Promoter trapping system to study embryogenesis.

Abstract

Promoter trapping is a particular gene trap strategy that represents a valuable tool for the discovery of specific cell-type markers. The principle is to generate a collection of transgenic lines with random insertions of a promoter-less reporter gene and to screen for specific reporter activity in the domain of interest. The use of beta-glucuronidase (GUS) as a reporter gene provides a simple and sensitive assay that allows identification of very restricted expression patterns and makes the promoter trap appropriate to study embryogenesis. Plant embryogenesis starts at the fertilization of the egg cell encapsulated in the maternal tissue and leads to the establishment of a new organism capable of an autonomous life. Uncovering genes specifically expressed in sub-domain of the embryo during its development represents a major technical challenge due, in part, to size and accessibility limitations. Promoter trapping approaches have been successfully used to overcome these problems. The trapped activity represents thereafter a useful genetic marker of the uncovered cell type, which is expected to reveal the properties of a specific promoter shedding light on a new gene function. In this chapter, protocols for examining and documenting GUS reporter gene activities in the embryo are described. Methods for the amplification of sequences flanking insertions and subsequent molecular and genetic characterization are provided.