Rescue Of Photoreceptors By BDNF Gene Transfer Using In Vivo Electroporation In The RCS Rat Of Retinitis Pigmentosa

PartⅠConstruction of BDNF-GFP fusion expression plasmid vector and study on its characteristicsObjective To construct a BDNF-GFP fusion expression plasmid and study its characteristics to supply basic evidences for its future's application in the study of gene therapy.Methods The BDNF CDNA was amplified by genomic PCR and then were inserted into pcDNA3.1/NT-GFP-TOPO plasmid and verified by sequencing.Result The construction of BDNF-GFP plasmid was verified correctly by sequencing。Conclusion The BDNF-GFP fusion expression plasmid vector was successfully constructed.Our study could provide a new method for the construction of target vector for gene therapy.PartⅡStudy of gene transfer into retinal pigment epithelium cells and photoreceptors using in vivo electroporationObjective To investigate the feasibility and the suitable electro-pulse model of transfecting DNA into retinal pigment epithelium(RPE) cells and photoreceptors(PRs) in vivo by electroporation method.Methods pEGFP-N1,the eukaryotic expressive plasmid of enhanced green fluorescent protein(EGFP) was injected into the subretinal space of Sprague—Dawley(SD) rats and serial electric pulses were applied by in vivo electroporation.Whole mounted retinas were made one week after introduction of plasmid and the fluorescence of EGFP was photographed by fluorescent microscope.The expression of EGFP mRNA and protein was detected by reverse transcription polymerase chain reaction technique(RT—PCR) and Western immunoblot analysis.Results EGFP gene was successfully transfected into RPE cells in vivo by a series of electric pulses,and the most suitable electro-pulse model is 10V/mm electric field strength with 99ms duration to each pulse,5 pulses with 0.5s interval each other.EGFP expression in PRs was not detected at present.Conclusion These findings indicate that electroporation is an effective method for gene delivery into RPE cells in vivo,but not for PRs.This new method may be an assistant tool for gene transfer into RPE cells in vivo,explore a potential treatment for retinitis pigmentosa.PartⅢRescue of photoreeeptors by BDNF gene transfer using in vivo electroporation in the RCS rat of retinitis pigmentosaObjective To investigate the feasibility of introducing brain-derived neurotrophic factor(BDNF) gene into retinal pigment epithelium(RPE) cells in vivo by electroporation and whether this method can rescue photoreceptors(PRs) of retinitis pigmentosa(RP) in Royal College Surgeons(RCS) rats.Methods The BDNF-GFP fusion eukaryotic expressing plasmid was constructed and then was subretinally or intravitreously injected into the eyes of Royal College Surgeons(RCS) rats followed by in vivo electroporation.The expression of BDNF mRNA and protein were detected by RT-PCR and Western immunoblot analysis.The number of surviving photorecptors was counted and the TdT-dUTP terminal nick-end labeling(TUNEL) method was used to detect the apoptosis of the retinal cells at different time points after introduction of BDNF plasmid.Results After the electrointroduction,the treated eyes showed a significantly higher rescue ratio and a lower number of TUNEL-positive photoreceptors than the control eyes at various time points. Conclusion These findings provide evidence that electroporation is an effective method for genes transfer into RPE cells and the rescue of PRs can be achieved by BDNF gene transfection with electroporation.