Creation of a Mouse Retinal Dp71 Vector and Expression In Vitro of a GFP-Dp71 Fusion Protein

Abstract

Purpose: : Dystrophin-Dp71 is a membrane-bound cytoskeletal protein that, in retina, is involved in glial and vascular functions. We have shown that Dp71 is involved in (i) the regulation of normal and pathological angiogenesis and (ii) the permeability of the Blood Retinal Barrier (see poster Giocanti A.) in mouse retina. Presently, only the partial sequence of the mouse Dp71 was reported. Here we characterized mouse retinal Dp71 at the molecular level to further develop Dp71 gene therapy tools.

Methods: : Total RNA was extracted from C57BL/6J adult mice retina. The amplicon from the RT-PCR was cloned into pcDNA3.1/NT-GFP-TOPO®vector. Restriction analysis and nucleotide automatic sequencing were performed for the Dp71 cDNA and for the Dp71 expression plasmid to compare both of them. Site-directed mutagenesis was made to correct the sequence of the Dp71 cloned. Expression into HEK-293 cell line was done. Western Blot was used to highlight the protein expression.

Results: : When RT-PCR was performed, a 1797 bp amplicon was obtained with the expected size of the Dp71 cDNA. The BamHI restriction pattern was obtained in accordance with the restriction pattern deduced from the sequence. The automatic sequencing was analyzed and compared to mouse Dp71 cDNA. Three mutations were identified and corrected by site-directed mutagenesis. Transfections with HEK-293 cells were performed with 90% efficiency. In Western Blot, a 100 kDa protein was obtained corresponding to the size of the GFP-Dp71 fusion protein.

Conclusions: : The complete sequencing of the retinal Dp71 cDNA has been performed successfully. The creation of a genetic tool to study the Dp71 expression was done with the necessary controls. Induction of the Dp71 expression in the retina would allow studying the molecular role of this protein in retinal angiogenesis and the BRB.

Keywords: gene/expression • gene transfer/gene therapy • genetics