Green fluorescent protein as a reporter for gene transfer studies in the cochlea

Anil K. Lalwani, Jay J. Han, Bong J. Walsh, Sergei Zolotukhin, Nicholas Muzyczka, Anand N. Mhatre

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

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 105 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-β-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.

Original languageEnglish (US)
Pages (from-to)139-147
Number of pages9
JournalHearing Research
Volume114
Issue number1-2
DOIs
StatePublished - Dec 1997

Fingerprint

Cochlea
Green Fluorescent Proteins
Reporter Genes
Dependovirus
Fluorescence
Organ of Corti
Transgenes
Guinea Pigs
Spiral Ligament of Cochlea
Galactosidases
Stria Vascularis
Genes
Spiral Ganglion
Gene Expression
Genetic Therapy
Virion
Safety
Polymerase Chain Reaction
Membranes

Keywords

  • Adeno-associated virus
  • Cochlea
  • Gene therapy
  • Gene transfer
  • Green fluorescent protein
  • Guinea pig

ASJC Scopus subject areas

  • Sensory Systems

Cite this

Lalwani, A. K., Han, J. J., Walsh, B. J., Zolotukhin, S., Muzyczka, N., & Mhatre, A. N. (1997). Green fluorescent protein as a reporter for gene transfer studies in the cochlea. Hearing Research, 114(1-2), 139-147. https://doi.org/10.1016/S0378-5955(97)00151-2

Green fluorescent protein as a reporter for gene transfer studies in the cochlea. / Lalwani, Anil K.; Han, Jay J.; Walsh, Bong J.; Zolotukhin, Sergei; Muzyczka, Nicholas; Mhatre, Anand N.

In: Hearing Research, Vol. 114, No. 1-2, 12.1997, p. 139-147.

Research output: Contribution to journalArticle

Lalwani, AK, Han, JJ, Walsh, BJ, Zolotukhin, S, Muzyczka, N & Mhatre, AN 1997, 'Green fluorescent protein as a reporter for gene transfer studies in the cochlea', Hearing Research, vol. 114, no. 1-2, pp. 139-147. https://doi.org/10.1016/S0378-5955(97)00151-2
Lalwani AK, Han JJ, Walsh BJ, Zolotukhin S, Muzyczka N, Mhatre AN. Green fluorescent protein as a reporter for gene transfer studies in the cochlea. Hearing Research. 1997 Dec;114(1-2):139-147. https://doi.org/10.1016/S0378-5955(97)00151-2
Lalwani, Anil K. ; Han, Jay J. ; Walsh, Bong J. ; Zolotukhin, Sergei ; Muzyczka, Nicholas ; Mhatre, Anand N. / Green fluorescent protein as a reporter for gene transfer studies in the cochlea. In: Hearing Research. 1997 ; Vol. 114, No. 1-2. pp. 139-147.
@article{51e0b8f8168142d9bab159ca9b3af9cf,
title = "Green fluorescent protein as a reporter for gene transfer studies in the cochlea",
abstract = "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 105 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-β-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.",
keywords = "Adeno-associated virus, Cochlea, Gene therapy, Gene transfer, Green fluorescent protein, Guinea pig",
author = "Lalwani, {Anil K.} and Han, {Jay J.} and Walsh, {Bong J.} and Sergei Zolotukhin and Nicholas Muzyczka and Mhatre, {Anand N.}",
year = "1997",
month = "12",
doi = "10.1016/S0378-5955(97)00151-2",
language = "English (US)",
volume = "114",
pages = "139--147",
journal = "Hearing Research",
issn = "0378-5955",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Green fluorescent protein as a reporter for gene transfer studies in the cochlea

AU - Lalwani, Anil K.

AU - Han, Jay J.

AU - Walsh, Bong J.

AU - Zolotukhin, Sergei

AU - Muzyczka, Nicholas

AU - Mhatre, Anand N.

PY - 1997/12

Y1 - 1997/12

N2 - 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 105 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-β-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.

AB - 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 105 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-β-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.

KW - Adeno-associated virus

KW - Cochlea

KW - Gene therapy

KW - Gene transfer

KW - Green fluorescent protein

KW - Guinea pig

UR - http://www.scopus.com/inward/record.url?scp=0031455805&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031455805&partnerID=8YFLogxK

U2 - 10.1016/S0378-5955(97)00151-2

DO - 10.1016/S0378-5955(97)00151-2

M3 - Article

C2 - 9447928

AN - SCOPUS:0031455805

VL - 114

SP - 139

EP - 147

JO - Hearing Research

JF - Hearing Research

SN - 0378-5955

IS - 1-2

ER -