Developing a technique for the targeted integration of genes in plants
(2008 - 2011) University of Karlsruhe, Botanical Institute, Chair of Molecular Biology and Plant BiochemistryTopic
When inserting new genes in plants, it is impossible to predict where in the genome integration will occur. As a result, the newly inserted gene could impair the functioning of other genes. Targeted insertion at known and well-defined sites in the genome could eliminate this uncertainty.
The aim of this project is to further develop a technique for the targeted integration of a transgene at any chosen site in the plant genome (gene targeting). This technique is based on homologous recombination , a naturally occurring mechanism whereby genetic material rearranges itself. As yet, gene targeting techniques developed for plants remain inefficient, i.e. the successful integration of the transgene at a specific site in the plant genome occurs too infrequently.
In the model plant Arabidopsis thaliana three genes (BRCA1, BRCA2 and BARD1) have been identified, the absence of which leads to significantly fewer homologous recombination events occurring. The aim of the experiment described here is to increase the number of homologous recombination events through the over-expression of this gene. If this is successful, tests will be conducted using two different gene targeting systems to determine whether this makes the technique more efficient.
Experiment description
In the first stage, Arabidopsis plants containing the three named genes will be transformed (cf. transformation), both individually and in different combinations. Over-expression of the genes will be induced by selecting suitable control elements (promoters ). Molecular-biological methods will be used to investigated the sites in the plant genome at which the genes have been inserted. A colour reaction will be used to identify which genes or gene combinations result in an increase in homologous recombination events. These plants or gene constructs will then be used in further experiments to investigate whether it is possible to improve the efficiency of the gene targeting technique in two different test systems.
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In the first system, the selected plants will be
transformed with a further gene construct which carries a reporter gene. The
reporter gene is flanked by sub-sequences of a gene which is found in the genome
of A. thaliana and is expressed only in the seed. The reporter gene will be
integrated at the site where this gene occurs in the plant genome via homologous
recombination and, as a result, it will also be expressed in the seed, and
nowhere else. If gene targeting is successful, the plants will produce red
fluorescent seeds, which can be very easily identified under a fluorescent
microscope and investigated further. |
The two parent strains from the precursor project will then be transformed with the gene constructs from the first part of the experiment, which successfully brought about an increase in homologous recombination events. Then the two strains will be crossed again to test whether the transgene is integrated more efficiently than in the precursor project.
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