Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

Method of somatic gene transfer into the brain of frogs

4.1. Optimisation of somatic gene transfer methods with the sleeping beauty system in Xenopus central nervous system. We have established that the somatic gene transfer techniques with DNA complexed with the polycationic polymer polyethylenimine for transfection into the CNS can be applied to the species under study: Xenopus tropicalis. Standard plasmids expressing different reporter genes (luciferase or GFP) have been injected with or without polyethylenimine (PEI), and the time course of expression and the relative yields calculated. The time course of expression in the CNS shows loss of transgene expression with time, which is one of the reasons we turned to the SB transposon system to obtain integration and long term expression of the transgene.

We addressed whether the SB transposon system could be adapted to improve the time course of expression by providing integration of the transgenes in the somatic tissue targeted. To this end we designed and constructed a series of plasmids that allowed us to follow transfection, as well as transposase-dependent excision and integration. These completed constructions include a plasmid with both dsRFP and GFP coding sequences (pdsRED/T-CMV-GFP) that enables the transposase-dependent excision step to be visualised by a switch in fluorescent emission. This plasmid is ready, and has been tested and shown to be functional in somatic gene transfer in the CNS . It was also been passed on to Partner 2 to be used in WP2 (germline transformation with SB transposase/transposon system). A second plasmid contains dsRFP and the ampORI selection gene (pdsRed /T-AmpORI) that can be exploited to follow integration of the gene of interest in the tissue transfected. A third plasmid (pT-CMV-luc) can be used to quantify expression of transgenes following different transfection conditions.

Each plasmid system was tested with the transposase expressed from a plamsid co-injected into the target tissue or with mRNA for the transposase. The latter method provides a more transitory expression of the enzyme and possibly avoid multiple insertions (that could be deleterious to the target cell).

The results show that transposase excision occurs but not integration. Thus, the method is not really better than standard plasmid constructs.

The method has been included in a paper submitted to Genesis.

Contatto

Barbara DEMENEIX, (Project Coordinator)
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