Seed-Specific Gene Activation Mediated by the Cre/lox Site-Specific Recombination System

Abstract

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][prime] 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.