Gene Transfer to Protoplasts: Transient Gene Expression Analysis

Abstract

While the aim of the first transformation experiments was to obtain stably transformed transgenic plants, direct DNA uptake by plant cells has also proven extremely useful as a means to analyze rapidly the expression and regulation of specific introduced genes via transient expression analysis. To address questions regarding mRNA stability or turnover, DNA constructs containing elements of interest in the 5’ and 3’ untranslated regions can be introduced and the fate of the mRNA produced can be followed. In addition, in vitro synthesized mRNA can be introduced directly to study, for example, the role of poly (A) tail length, a feature that cannot be controlled otherwise (Gallie et al. 1989, 1991). To gain information about the expression, regulation, and tissue specificity of specific genes for which analysis of the gene product is not easy, regulatory regions of a gene of interest such as a promoter, enhancer, leader, 3’ untranslated regions, or intron can be fused with a reporter gene. Reporter genes in flexible cloning cassettes with an appropriate promoter exist for firefly luciferase (luc), E. coli (3glucuronidase (GUS) and chloramphenicol acetyltransferse (CAT). These vectors produce enzymes that are easily assayable within hours of transformation (Fromm et al. 1987; Jefferson 1987; Luehrsen et al. 1992).