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Transactivation Lines in Rice

Evaluation of insertional mutants and development of effective transactivator platform for FTO and co-ordinate gene expression

by Marie Connett Porceddu, Thach Tran, Richard Jefferson and Andrzej Kilian, CAMBIA.

 August 2006

RIRDC Publication No 06/095   RIRDC Project No CMB-2A

Executive Summary
This project aimed to help secure freedom to operate for the Australian rice industry in the biotechnology era. The initial focus of this project was in a gene discovery mechanism, hoped to lead to the discovery of rice promoters with useful gene expression patterns, and mutants that might have improved gene expression properties. However, the rice genomic sequencing efforts external to Australia, rapidly accelerated during the course of the project, changed the terms of reference. What was needed was no longer large numbers of mutations, but
 
• Ways to work around the large number of patent applications that followed the release of genomic sequence, to the extent that licenses to such patents may not be available or may block access to external markets.
• Ways to understand and direct gene expression now that we understand the genome to be more complex and studies of single gene insertions effects to be too simplistic.


At the end of the project, these concerns have both been addressed. A patent work-around was developed, a strategy to co-transform or cross the vector containing the open reading frame for the transcriptional activator, and the vector containing the DNA-binding activation site, so that the insertions would be unlinked. In patent claims over the use of promoters, often what is claimed is a construct that comprises the promoter “operably linked” to a gene of interest. In transactivation, the promoter of interest is not linked to a gene of interest, but to a transcriptional activator. We also constructed a set of new transactivator cassettes, through identification of an activation domain and a DNA binding domain that work better in plants and are less clearly dominated by IP rights.

Lines that express this transactivator in a defined pattern are determined by the enhancers or promoters near the site of insertion. Genes that it is desired to switch on can be placed into cassettes with a minimal promoter and an activation sequence for the transactivator. When these cassettes are transformed into plants containing or crossed with the pattern lines, the gene(s) of interest will be brought into the expression pattern shown by the reporter genes in a highly controlled useful manner without the need to define, analyse, and clone new promoters to drive the gene(s) of interest. Thus:

• the expression of multiple genes encoding different enzymes in a multi-step pathway, such as the starch biosynthesis pathway, can be controlled to occur at the same developmental stages and in the same amounts in the plant.
• The expression of down-regulation constructs for multiple steps in a pathway can similarly be coordinately regulated
• The expression of either type of construct can be monitored by reporter gene expression.


“Freedom to co-operate” considerations are equally important with “Freedom to operate” and technical evaluation. The new transactivator is now available to the rice industry, but even more useful, it is presented as a capability to use it, together with the accumulated know-how, in a form that enables ready collaboration with other researchers and capture of the data they collect and the improvements they make, in the BioForge project (www.bioforge.net) being developed since 2005 by CAMBIA with US-sourced funding from the Rockefeller Foundation. Facilitating joint improvement and leverage, all technology presented on the BioForge is available to other researchers in the public and private sectors only if they agree to the collaborative terms of the BiOS license (www.bioslicense.net).

This unique legal instrument developed by CAMBIA provides for norms of sharing, as well as legally binding covenants that protect those who have invested in a technology, such as RIRDC, from being subject to the situation of inability to use valuable improvements to the technology.

A summary of the BiOS License conditions follows: In return for the benefits of the technologies, a licensee institution agrees to allow and encourage its employees and students to post on the website any improvements made to the technologies and safety information relevant to use of the technology and potential regulatory approval of products embodying it, and agrees not to assert any intellectual property rights to the improvements and information against other licensees.

 


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Last updated: August 2006    Copyright RIRDC
http://www.rirdc.gov.au/reports/RIC/06-095sum.html