Dr. Sebastian Oltean, Institute of Biomedical & Clinical Sciences, University of Exeter Medical School, UK.

Speech Title: Use of splicing-sensitive fluorescent reporters to screen for modulators of VEGF-A splicing in vitro and to understand splicing regulation in vivo in transgenic mice

Abstract: Through alternative splicing of the terminal exon in the VEGF-A gene, two functionally distinct isoform families are generated. Use of the canonical proximal 5' splice site results in the expression of the pro-angiogenic VEGF-Axxx isoforms, whereas use of the distal 3' splice site results in anti-angiogenic VEGF-Axxxb expression.

A bichromatic splicing-sensitive fluorescent reporter (SSFR) was engineered to mimic VEGF-A exon 8 splicing. The reporter (termed VEGF8ab) consists of the endogenous VEGF-A intron 7 and required parts of exon 8, followed by the coding sequences for two fluorescent proteins. Depending on which 3' splice site is used, a different fluorescent protein is expressed - RFP denotes the pro-angiogenic VEGF-Axxx isoforms, and GFP denotes the anti-angiogenic VEGF-Axxxb isoforms. This splicing reporter allows VEGF-A alternative splicing to be studied both in vitro and in vivo.

Following successful validation, a screen of 1280 pharmacologically active compounds (LOPAC library) was performed using several screening methods to identify nine hit compounds that switched the alterative splicing pattern, therefore decreasing the RFP/GFP ratio. Further experiments were carried out to confirm that several of the compounds switched the endogenous splicing pattern of VEGF-A. Additionally, treatment of tumors and Matrigel plugs with these compounds resulted in decreased tumor growth and vessel density due to anti-angiogenic activity.

We have also engineered a transgenic mouse harbouring the VEGF8ab. Preliminary data from this mouse model confirms recent findings regarding VEGF-A splicing; e.g. in skeletal muscle it has been reported that VEGF-A165b levels are high and indeed we see an abundance of GFP in skeletal muscle. This mouse model is further used to assess the effect of splicing compounds in vivo.

Data will be presented to show the utility of this new technology in studying regulation of alternative splicing in vitro and in vivo, applicable to virtually any splice event.

Keywords: alternative splicing, splicing reporters, VEGF-A