December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
High Efficiency Transfection of Rat Retinal Ganglion Cells by a Modified Adeno-associated Virus Incorporating the Woodchuck Hepatitis Post-transcriptional Regulatory Element
Author Affiliations & Notes
  • KR Martin
    Wilmer Eye Institute Johns Hopkins Hospital Baltimore MD
  • RL Klein
    Department of Pharmacology and Therapeutics University of Florida Gainsville FL
  • H Levkovitch-Verbin
    Wilmer Eye Institute Johns Hopkins Hospital Baltimore MD
  • L Baumrind
    Wilmer Eye Institute Johns Hopkins Hospital Baltimore MD
  • D Valenta
    Wilmer Eye Institute Johns Hopkins Hospital Baltimore MD
  • M Pease
    Wilmer Eye Institute Johns Hopkins Hospital Baltimore MD
  • HA Quigley
    Wilmer Eye Institute Johns Hopkins Hospital Baltimore MD
  • Footnotes
    Commercial Relationships   K.R. Martin, None; R.L. Klein, None; H. Levkovitch-Verbin, None; L. Baumrind, None; D. Valenta, None; M. Pease, None; H.A. Quigley, None. Grant Identification: Frost Trust (UK), Oxford University (UK), Alzheimer's Association, EY02120/01765
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 2888. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      KR Martin, RL Klein, H Levkovitch-Verbin, L Baumrind, D Valenta, M Pease, HA Quigley; High Efficiency Transfection of Rat Retinal Ganglion Cells by a Modified Adeno-associated Virus Incorporating the Woodchuck Hepatitis Post-transcriptional Regulatory Element . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2888.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Abstract: : Purpose: Integration of novel gene sequences into retinal ganglion cells (RGC) is a promising approach in the study of degenerative RGC diseases such as glaucoma. Previously published studies, however, have often had low or unquantified RGC transfection rates. Our aim was to develop a modified adeno-associated virus (AAV) vector which could reliably and specifically transfect a high proportion of RGC following a single intravitreal injection. Methods: A modified AAV carrying the gene for green fluorescent protein (GFP) under the control of a chicken beta-actin promoter and incorporating the woodchuck hepatitis post-transcriptional regulatory element (WPRE) was constructed. One eye of 20 adult Wistar rats received a single intravitreal injection of 2ml viral stock (2x1012 particles/ml). Two weeks after injection, retinal wholemounts (n=13 rats) and histological sections (n=7 rats) were prepared. The density of GFP-positive cells was calculated in digital images of randomly sampled wholemount fields under fluorescence microscopy. Measurement of retinal wholemount area allowed estimation of the total number of transfected cells per retina. This value was compared to the number of axons in optic nerve cross sections and to the number of RGC back-labelled 10 days after fluorogold injection to the superior colliculus. Results: The mean density of GFP-positive cells two weeks after AAV injection was 1828±299 cells/mm2, equivalent to 72274±11814 cells/retina (mean±SD, n=13). Almost all GFP-transfected cells seen in retinal cross sections were localized to the RGC layer, except in the immediate vicinity of the injection site where some cells in deeper retinal layers were labelled. Uninterrupted axonal projections of many GFP-positive cells could be traced from the retinal periphery to the optic nerve head in directly visualized retinal wholemounts. The mean number of GFP-transfected cells was 84.5±13.8% of the mean number of axons counted in normal rat optic nerve cross sections and 74.7±12.2% of the number of retinal cells back-labelled following fluorogold injection to the rat superior colliculus. Conclusions: Efficient transfection of a high proportion of RGC can be obtained after a single intravitreal injection of an AAV incorporating the woodchuck hepatitis post-transcriptional regulatory element.

Keywords: 419 gene transfer/gene therapy • 415 ganglion cells • 316 animal model 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×