Transgene Silencing: New Insight to Genetic Mechanisms

Texas A&M Research Foundation, College Station TX

Investigators

Abstract

Transgene silencing is the unexpected lack of expression of a gene introduced into an organism through recombinant engineering. Many lines of evidence have led to the realization that gene silencing reflects a series of strategies that protect the genetic information present in an organism from interference by extraneous genetic information. Some of these strategies are related to well-known defense mechanisms that provide protection against infectious organisms, but others have not previously been recognized and their mechanisms are only just now being investigated. These considerations are reflected in the goal of this project, which is to identify genes involved in transgene silencing mechanisms and to characterize their operation in normal development. In addition to providing new insight to genetic control systems of transgene expression, this understanding will lead to the strategic design of future transgene constructs that will not be sensed by the recipient genome as being alien and thus be expressed more reliably. A premise for the experiments to be undertaken is that inactivation of genes that are responsible for the silencing machinery will re-activate the silenced gene. Three different sets of rice lines, in which a transgene is transcriptionally silenced, have been developed and characterized that are eminently suitable for reactivation studies. However, because of its rapid growth cycle and its fully-sequenced genome, Arabidopsis will be used as a model plant to expedite selection of gene targets for rice. Inactivation of the host silencing system(s) will be accomplished primarily by an RNA interference (RNAi) approach in which double-stranded RNA corresponding to the gene target is expressed. Initial gene targets include rice methyltransferase, chromo domain-containing genes and histone deacetylase genes. This is because methylation and alteration of chromatin structure is known to be associated with many gene silencing events. Once a target gene yields an interesting knock-out (silencing inactivation) phenotype in Arabidopsis, existing databases will be searched for similar genes in rice. RNAi-based knock-out constructs of these genes will be made and their ability to restore transgene expression in the silenced rice lines investigated to evaluate the role of the knocked-out gene as being one responsible for silencing.