Analyse of the characteristics of phiC31 integrase-mediated minicircle generation

Several platforms exist for controlled genomic engineering (e.g. polymerases, restriction enzymes, site-specific recombinases, transposases, zink finger nucleases, TALENs etc.).Site-specific recombination systems became an efficient tool for the precise in vivo modification of eukaryotic genomes. They are mainly used in animal systems and mammalian cells, but receive increasing attention in plants. In the case of directly repeated recognition sites, the recombination reaction results in the loss of the intervening DNA and a circular molecule is formed from the excised fragment. Constructs very similar to the circular DNA generated by site-specific recombinases are applied in the field of minicircles to enhance expression of the gene-of-interest and obtain high yields of various recombinant proteins. The use of small episomal circles, lacking redundant bacterial sequences, may minimize the risk of DNA methylation leading to epigenetic changes, and hereby silencing of the gene-of-interest. Strikingly, the episomal circle might exist autonomously in the cell after the recombination reaction, and is not necessarely subjected to an immediate cellular degradation after its emergence. However, little is known about its in vivo behavior in plant systems. The currently available information is insufficient to determine the fate of the removed nuclear sequence after recombinase-mediated excision. This issue is important because a persistence of the excised products in somatic cells may pose biosafety concerns. If the episomal circle proves to be stable and persistent in dividing cells, one cannot rule out the possibility that it is passed on to successive generations of the organism. In order to reveal the potential of plant minicircles to enhance target gene expression and to reach a high practicability in applied systems, it is necessary to analyze the associated mechanisms, such as episomal excision circle formation, stability and monitoring its transgene expression. Therefore, to understand the consequences of the site-specific recombination reaction, the proposed project reveals a strong indication for extensive further analyses of recombinase-mediated site-specific excision. This can be achieved via tracing the episomal circle generated by the phiC31 integrase in plants. According to preliminary experiments, the target chromosomal segment removed by a site- specific recombinase, the phiC31 integrase, remains stable in barley. Transgenic Arabidopsis thaliana and barley (Hordeum vulgare) cv. Golden Promise will be used as model systems to achieve the goals using molecular methods and two fluorescent reporter proteins for tracking the process of phiC31 integrase-mediated site-specific excision, determine the exact timepoint of the minicircle formation and monitor its fate in the cells. The results will be of great value for the fields of biosafety and the possible application of the system for enhanced recombinant protein expression.

Several platforms exist for controlled genomic engineering (e.g. polymerases, restriction enzymes, site-specific recombinases, transposases, zink finger nucleases, TALENs etc.). Site-specific recombination systems became an efficient tool for the precise in vivo modification of eukaryotic genomes. They are mainly used in animal systems and mammalian cells, but receive increasing attention in plants. In the case of directly repeated recognition sites, the recombination reaction results in the loss of the intervening DNA and a circular molecule is formed from the excised fragment. Constructs very similar to the circular DNA generated by site-specific recombinases are applied in the field of minicircles to enhance expression of the gene-of-interest and obtain high yields of various recombinant proteins. The use of small episomal circles, lacking redundant bacterial sequences, may minimize the risk of DNA methylation leading to epigenetic changes, and hereby silencing of the gene-of-interest. Strikingly, the episomal circle might exist autonomously in the cell after the recombination reaction, and is not necessarely subjected to an immediate cellular degradation after its emergence. However, little is known about its 'in vivo' behavior in plant systems and the fate of the removed nuclear sequence after recombinase-mediated excision. This issue is important because a persistence of the excised products in somatic cells may pose biosafety concerns. If the episomal circle proves to be stable and persistent in dividing cells, one cannot rule out the possibility that it is passed on to successive generations of the organism. In order to reveal the potential of plant minicircles to enhance target gene expression and to reach a high practicability in applied systems, it is necessary to analyze the associated mechanisms, such as episomal excision circle formation, stability and monitoring its transgene expression. Therefore, to understand the consequences of the site-specific recombination reaction, the proposed project aimed at extensive analyses of recombinase-mediated site-specific excision. This can be achieved via tracing the episomal circle generated by the phiC31 integrase in plants. According to the study, the target chromosomal segment removed by a site-specific recombinase, the phiC31 integrase, remains stable in the two model systems: Arabidopsis thaliana and barley (Hordeum vulgare). The goal was achieved using molecular methods and two fluorescent reporter proteins for tracking the process of phiC31 integrase-mediated site-specific excision. The timepoint of the minicircle formation was determined and its fate in the cells was monitored. The results will be of great value for the fields of biosafety and the possible application of the system for enhanced recombinant protein expression.