De invloed van dubbelstrengige breuken en het type doelwitcel op de efficiëntie van "gene targeting" in planten.

In this work a number of biotechnological issues regarding the generation and use of transgenic plants were addressed.

First, we isolated promoter sequences form Arabidopsis thaliana genes which are active in germline cells in a broad spectrum of developmental stages: shoot apical meristem, flower meristem and meiosis. We then investigated to what extent these promoters can regulate expression of the Cre site-specific recombinase in order to result in excision of a DNA sequence flanked by tandem oriented lox sites. The selected germline promoters are involved in different developmental cues including early stem cell identity (CLAVATA3), flower meristem identity (LEAFY, APETALA1), floral organ identity (AGAMOUS), and meiosis (SOLO DANCERS, DMC1, SWITCH1), and the functionality of each promoter in the germline of primary transformants was evaluated by analyzing the presence of the recombined lox cassette in T2 progeny. We showed for 5 out of these 7 promoters that efficient Cre-mediated recombination does indeed occur and that the recombination takes place at some point during germline development. These data are significant because they enabled us to select a number of germline promoters for two novel applications.

A first application in which these germline promoters were used represents a novel gene targeting approach. Gene targeting is the ability to perform any kind of targeted genome modifications. This application represents a powerful biotechnological tool because it allows precise introduction of modifications or deletions of any chosen genomic sequence. In plants, the development of an efficient gene targeting method has remained elusive for years (except in the moss Physcomitrella patens). In contrast, in other species gene targeting is feasible but seems to be restricted to specific cell types (such as embryonic stem cells (ES) in mouse, Drosophila germline cells and chicken DT40 cells). These findings seem to indicate that the species or cell type can greatly influence gene targeting efficiencies and forms the basis of our experimental rationale.

Our initial strategy consisted in activating the gene targeting system during meiosis of Arabidopsis thaliana. This was achieved by Cre-mediated release of a circular targeting DNA, which was placed between two tandem oriented lox sites, during prophase I of meiosis. This targeting sequence shares homology with a target sequence which contains a defective reporter gene. When homologous recombination between the target and targeting sequence occurs the defective reporter gene is restored. We were able to show that Cre-mediated in planta presentation of the targeting sequence can in some cases result in the restoration reporter gene. However, our results also seem to indicate that reporter gene is restored on the targeting DNA and subsequently integrated elsewhere in the genome; a phenomenon termed ectopic gene targeting.

The germline promoters were also applied in another novel technology which enables the generation of transgenic plants devoid of selectable marker genes. The generation of transgenic plants through the existing transformation techniques is relatively inefficient; marker genes are therefore required for efficient selection of transformed plant cells mostly through resistance against either an antibiotic or an herbicide. The presence of the selectable marker gene is generally unwanted after it served its purpose because it can affect expression of other transgenes and requires the use of other marker genes in subsequent transformation steps. Our system is based on Germline-Specific Auto-excision (GSA) of the selectable marker gene, and consists of Cre-mediated removal during germline development of a lox cassette containing a selectable marker gene. We tested both in Arabidopsis and Nicotiana tabacum the efficiency of the GSA vector, with the germline promoters of the Arabidopsis genes regulating cre expression. According to our results, marker-free transgenic plants can be obtained when the promoters of the LFY, AP1, SDS and DMC1 genes are used in the GSA vector. In addition we show that these promoters give similar results with regard to marker-free progeny in N. tabacum.



In conclusion, promoters which can achieve germline directed Cre-lox recombination in plants clearly provide an interesting improvement to the existing plant biotechnological tools. The versatility of this tool has been demonstrated in this work by its use in distinct applications which can represent a further refinement of the current genome modifying technologies in plant.