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Evaluation of AAV-mediated sox2 overexpression on adult human RPE cells

Session Details

Session Title: New Drug Treatment and Technology II

Session Date/Time: Sunday 20/09/2015 | 09:00-10:30

Paper Time: 09:32

Venue: Calliope

First Author: : S.Samiee IRAN, ISLAMIC REPUBLIC OF

Co Author(s): :    A. Etemadzadeh   Z. Soheili   R. Ezzati   H. Ahmadieh   E. Ranaei  

Abstract Details

PURPOSE:Retinal pigment epithelium (RPE) is a monolayer of cells underlying and supporting the neural retina. Developmentally, RPE and neural retina originate from the same structure, the optic vesicle. Sox2 an HMG box transcription factor, plays an essential role in the eye development. The aim of this study was the evaluation of recombinant AAV-2 containing sox2 competence in reprograming adult human RPE.

Setting:

The aim of this study was the evaluation of recombinant AAV-2 containing sox2 competence in reprograming adult human RPE.

Methods:

Coding region of sox2 gene was cloned into pAAV-MCS vector. Validity of cloning was approved by colony PCR, digestion and sequencing. To detect gene expression, a 1.3kb IRES-EGFP segment as a reporter gene was ligated into the expression vector. Bacteria transformed by ligation products were confirmed by quick check extraction. Digestion, PCR and sequencing methods were done to verify the final expression vector. pAAV-lacZ was used as a reporter vector to optimize calcium phosphate method for transfection. HEK293T cells which were co-transfected with recombinant vector containing SOX2-IRES-EGFP, pAAV-RC and pHelper. Recombinant viruses were harvested after 72 hours. To determine virus titration, X-Gal staining, flow cytometry and absolute real-time polymerase chain reaction (PCR) were used. Cultured RPE cells obtained from adult human cadaver globes were infected by viruses. Real-time PCR and immune-cytochemical(ICC) analysis were utilized to evaluate the expression of sox2 and retinal cells' markers.

Results:

Titration of viral particles revealed a concentration of 1.8×106 infectious viruses/mL of primary preparations. Suspense infection method was selected as the best way of RPE infection. Real time PCR showed Nestinover-expression followed by sox2 significant over-expression after repression of cell proliferation by camptothecin. In addition, changes in pax6 expression were detected. ICC represented Nestin protein expression as neural progenitor marker in cells that were infected by AAV-SOX2 viruses. . The expression of Thy1and Rho protein was also detected in ICC.

Conclusions:

The presented data implied that sox2 over-expression induced adult human RPE cells retro differentiation.

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