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FP6 Priority: 1 (LSH-2002-1.2.0-1)

Projects Acronym: GENINTEG  (Mechanisms of gene integration)

Start and Ending date: 01.01.2004-2008 (48 month)

Budget: 3,602,663 (Requested EC contribution: 1,846,561)

Name Coordinator: Jean-Marie Buerstedde

Full Address:

Institute of Molecular Radiobiology, GSF

Ingolstädter Landstr. 1

D-85764 Neuherberg, Munich

Tel: 49 89 3187 2871

Fax: 49 89 3187 4093

Full Title: Controlled gene integration: a requisite for genome analysis and gene therapy

Contract number: FP6-503303 (LSHG-CT-2003-503303)

SPECIFIC TARGETED RESEARCH OR INNOVATION PROJECT (STREP)

1. Project summary 

Despite the enormous potential of using transgenesis for gene function analysis and gene therapy, little is known about the mechanism of gene integration in eukaryotic cells. Transgene integration into the chromosomes of living cells can occur either randomly or targeted by homologous recombination. The later type of integration is the most useful, because it allows precisely deleting or modifying sequences at defined chromosomal positions.

A consortium is formed to study gene integration by recombination in a number of different model organisms. As DNA repair is conserved during evolution, a comparative genomics approach is proposed to discover evolutionary conserved principles of gene integration. An explicit goal is to understand why targeted integration occurs efficiently in some eukaryotic cells, but not in others. One explanation to be investigated is that the ratio of targeted to random integration reflects the balance between DNA double-strand break repair by homologous recombination and end-joining. Other areas of research will relate to site-specific integration by AdenoAssociatedViruses, T-DNA elements and serine recombinases.

The insight into the mechanism of gene integration will be used to increase the efficiency of targeted gene integration in cells, where targeted integration events are currently hard to detect. This might be achieved either by modification of the gene constructs or their transfer into the cell nucleus or by regulating the expression of transacting recombination factors. As gene targeting is the only way to precisely modify the genetic blueprint of living cells in vivo, improvements of this technique offer immense opportunities not only for basic research, but also for biotechnology and gene therapy. This ranges from fine-tuning protein production in plants and bio-reactors to gene therapy for autosomal dominant defects. A SME company is included in the project to capture these opportunities and reinforce the competitiveness within the Community.

2. Project objective(s)

The main objective is to understand and enhance gene integration through interdisciplinary, comparative genome analysis in different model organisms.  As DNA structure and DNA repair are conserved during evolution, the gained knowledge and resources will improve gene integration across plant and animal species and facilitate large-scale gene function analysis and transgene expression for biotech and medical applications.

1) Better insight into the genetics, the regulation and the mechanism of homologous recombination

2) New protocols to increase targeted gene integration in primary cells and cell lines either by modification of gene constructs or the gene delivery mode

3) Adaptation of existing site specific recombination system for safe and stable gene expression and long range chromosome engineering

4) Use of improved gene integration for gene function analysis

5) Exploitation of the generated knowledge and resources for commercial application through the protection of intellectual property and product development

6) New protocols for transgenesis of whole organisms

The consortium will profit from the combined expertise of all partners which have been studying chromosomal recombination and DNA integration in a different model organisms.  Eukaryotic cells have the potential to integrate transfected DNA either at random chromosomal sites or targeted by homologous recombination. A central question is why gene constructs integrate efficiently by homologous recombination only in some eukaryotes like the yeast S. cerevisiae, the moss Physcomitrella patens and the chicken B cell line DT40. Are species and cell type specific recombination proteins responsible for this difference or is it caused by an overall shift in the balance of double-strand break (DSB) repair by homologous recombination? An answer to this question is of considerable interest, since it would reveal how to elevate the low targeted integration efficiencies in many species. A promising break-through is the recent demonstration that targeted gene knock-out can be achieved in Drosophila melanogaster by the intracellular release of open ended gene constructs. It is planned to further improve this technique and transfer it to other model organisms like for example Arabidopsis which up to now integrate genes almost exclusively at random chromosomal positions.

Apart from homologous recombination, site-specific recombination can be used to control gene integration with clear benefits for biotechnology and biomedical applications. First, the relationship between T-DNA element integration and DSB repair will be investigated in Arabidopsis to develop a robust technique for single copy integration at a defined site. As targeted integration is not feasible in many plants species, site-specific integration is an attractive option to achieve efficient and stable gene expression. In a similar manner, the site-specific integration features of Adeno-Associated Viruses will be adapted for biopharmaceutical production in CHO cells. It is also planned to adapt the tightly controlled bacteriophage serine recombinases for the engineering of mammalian mini-chromosomes as well as for transgenesis in C. elegans. A need for transgene integration into a defined site also exists in Drosophila. 

The know-how and technology to be developed in the consortium will open new opportunities to use transgenesis for industrial applications. Two industrial partners are included in the consortium to assure that valuable intellectual property is identified, protected and commercially exploited.

Jean-Marie Buerstedde

GSF-Research Center for Health and Environment

Institute of Molecular Radiobiology, GSF

Ingolstädter Landstr. 1

85764 Neuherberg, Munich

William Brown

University of Nottingham

University Park

Nottingham NG7 2RD

Prof. A. Depicker

Ghent University

Vakgroep Moleculaire Genetica

Ledeganckstraat 35,

9000 Gent

Plant Systems Biology VIB Department

Technologiepark 927

9052 Gent , Belgium

Dr. Francis FABRE

Commissariat a l'Energie Atomique

DSV/DRR. Bât.05

BP 6.

92265 FONTENAY-AUX-ROSES CEDEX, France

Prof. Dr. Martin Fussenegger

Professor of Biotechnology and Bioengineering                      

Institute for Chemical and Bio-Engineering (ICB)                       

Wolfgang-Pauli-Strasse 10                                                      

ETH Hoenggerberg, HCI F115                                                             

CH-8093 Zurich, Switzerland                                                                

Andrzej Kierzek

School of Biomedical and Molecular Sciences

University of Surrey

Guildford. GU2 7XH

Bernd Reiss, Ph.D.

Max-Planck-Institut fuer Zuechtungsforschung

Carl von Linne Weg 10

50829 Koeln, Germany

Walter Schaffner

Institute of Molecular Biology

University of Zurich

Winterthurer Str. 190

CH-8057 Zurich, Switzerland

Projects Web Link: http://pheasant.gsf.de/DEPARTMENT

This Form was completed by – Name: Randolph B Caldwell

Affiliation to the project: Scientist in the lab of J-M Buerstedde

Submission Date of this form: 14MAR05