Green fluorescent protein as a reporter for gene transfer studies in the cochlea.
This study examined the 'humanized, red-shifted' version of the jellyfish Aequorea victoria green fluorescent protein (hrGFP) as a novel reporter for in vivo gene transfer studies in the cochlea using adeno-associated virus (AAV) vectors. Approximately 10(5) AAV vectors containing the hrGFP reporter gene were infused over 2 days or 1 week into the cochlea of the guinea pig via an osmotic minipump. Saline infused, non-infused, as well as AAV-beta-galactosidase infused guinea pigs served as the negative controls. The hrGFP transgene expression was detected as moderate intensity fluorescence easily distinguished from the background. Increased fluorescence was seen in the spiral ganglion, spiral ligament, spiral limbus, organ of Corti, and Reissner's membrane of the AAV-hrGFP infused animals. Control animals showed minimal fluorescence throughout the cochlea. Comparison of the 2 day and 1 week AAV-hrGFP infused animals showed qualitatively increased fluorescence in the 2 day animals. Background autofluorescence in the stria vascularis was noted in both the experimental and the control animals. In addition, fluorescence was detected in the contralateral cochlea of the AAV-hrGFP infused animals. Subsequent PCR analysis confirmed the presence of viral particles in the AAV-hrGFP infused cochlea as well as in the brain and the contralateral cochlea. This finding has important implications for the eventual implementation of cochlear gene therapy. The results not only reinforce the need to assess the introduction and expression of foreign genes in the target cochlea but also consider issues of viral spread, safety, and modes of gene delivery. This study establishes hrGFP as an effective reporter of gene transfer and transgene expression in the cochlea. GFP's small gene size, stability, ease of detection, and potential for diverse biological applications will be invaluable for a variety of future gene transfer and expression studies in the cochlea.
Lalwani
A K
AK
Epstein Laboratories, Department of Otolaryngology-Head and Neck Surgery, University of California San Francisco, 94117, USA. lalwani@itsa.ucsf.edu
Han
J J
JJ
Walsh
B J
BJ
Zolotukhin
S
S
Muzyczka
N
N
Mhatre
A N
AN
D000818:Animals; D003051:Cochlea / Q000166:cytology / Q000378:metabolism*; D017931:DNA Primers / Q000032:analysis; D000229:Dependovirus / Q000201:enzymology / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006168:Guinea Pigs; D008164:Luminescent Proteins / Q000235:genetics*; D008297:Male; D008856:Microscopy, Fluorescence; D009925:Organ of Corti / Q000378:metabolism; D016133:Polymerase Chain Reaction; D037401:Scyphozoa; D013136:Spiral Ganglion / Q000378:metabolism
D017931:DNA Primers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins