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Cellular and animal models for the investigation of gene regulation in the native environment of the living cell nucleus

Objective

Objectives
: A specific limiting factor in designing genetically based intervention therapies is that the optimal target molecule or multi-molecular complex is not usually known for a given disease situation because very little information is available regarding the components involved in the control of gene expression and their mode of interaction in what is now known to be a very sophisticated spatial and temporal context within the nucleus. For this purpose most of the currently used methods are clearly insufficient as they rely on the permeabilisation of cells and the fractionation of nuclei which are likely to introduce important deviations from the actual native state in the dynamic, living cell. The participants therefore propose to use and further develop state of the art methods allowing the direct spatial and temporal visualisation of macromolecular interactions relevant to gene control in vivo.

At present gene intervention therapies aimed to delay or correct cell transformation have been significantly limited by several basic problems of gene function and regulation, which remain poorly understood. As a result, gene transfer technologies have failed to achieve a therapeutic value in the majority of cases. Clearly, more attention needs to be focused on basic questions about how cells utilise genetic information to mediate biological processes such as gene expression, replication, repair and recombination. A specific limiting factor in designing genetically based intervention therapies is that the optimal target molecule or multi-molecular complex is not usually known for a given disease situation because very little information is available regarding the components involved in the control of gene expression and their mode of interaction in what is now known to be a very sophisticated spatial and temporal context within the nucleus. For this purpose most of the currently used methods are clearly insufficient as they rely on the permeabilisation of cells and the fractionation of nuclei, which are likely to introduce important deviations from the actual native state in the dynamic, living cell.

We therefore propose to use and further develop state of the art methods allowing the direct spatial and temporal visualisation of macromolecular interactions relevant to gene control in vivo. This will lead to the development of a number of cellular and animal model systems which will not only provide greater insight into basic macromolecular interactions in vivo, but will also act as test systems for disease processes and the design of therapeutic strategies.

The stages of the proposal are:
1. The development of ultra high sensitive technology to detect a minimal number of fluorescent molecules in a single living cell;
2. The development and improvement of methods that allow an efficient delivery of antisense probes into cells (e.g. microinjection, electroporation, particle bombardment);
3. Construction of a cellular model that will allow the continuous assessment of the stage of the cell cycle by direct microscopic observation on a single, living cell basis and which may be used to study the interactions of genes predisposing to cancer;
4. The development of methods to visualise the biosynthesis of mRNA molecules in living cells and the extension of this approach to screen accessible target sites on nuclear RNA within an antisense oligonucleotide protocol;
5. Address issues regarding "chromatin ilexibility".

The mobility of genes in the nucleus will be determined throughout the cell cycle in order to assess the probability of collision between genes involved in translocations causing leukaemia. Novel information will also be provided for the design of optimal LCR-based expression vectors by identifying the minimal distance required between the LCR and the promoter of choice for maximum activation, using the human beta-globin gene locus as a model system in the first instance. Electron microscopic in situ hybridisation methods will be developed to visualise direct interactions between distantly located genetic control elements in particular LCRs and their cognate promoter(s). This technology will be developed using the globin LCR as a model system and then extended to localise the LCR within the human desmin gene domain. These objectives fit directly with area 4.1.4 of the Biomed 2 programme.

Keywords: transgenic mouse models; gene regulation; gene interactions; cell cycle; antisense oligonucleotides. 04 04

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

FUNDACAO DA UNIVERSIDADE DE LISBOA
Address
Avenida Prof. Egas Moniz
1649-028 Lisboa
Portugal

Participants (3)

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Germany
Address
Meyerhofstrasse 1
69117 Heidelberg
Erasmus Universiteit Rotterdam
Netherlands
Address
50,Dr. Molewaterplein
3000 DR Rotterdam
United Medical and Dental Schools of Guy's and St Thomas's Hospitals
United Kingdom
Address
London Bridge
SE1 9RT London