Adeno-associated viral mediated transfer of the Lac Z reporter gene in the optic nerve and retina of the guinea pig

J. Guy, X. Qi, N. Muzyczka, W. W. Hauswirth

Research output: Contribution to journalArticle

Abstract

Purpose - To determine the feasibility of introducing foreign genes into the optic nerve. Methods - We linked a CMV promoter to a Lac Z-SV40 polyA reporter gene that was inserted into a bacterial plasmid containing adeno-associated virus (AAV) terminal repeat sequences. The promoter/gene construct was packaged into AAV particles by transfection into a human cell line that had also been infected with a temperature-sensitive helper virus and transfected with a replication-defective AAV packaging plasmid. The resultant AAV-packaged virus, CMV-Lac Z, was harvested and concentrated. We injected 100 μl containing ∼ 108 pfu of the viral-encapsulated cDNA gene constructs into the vitreous cavity of 10 eyes of 10 anesthetized strain-13 guinea pigs. We performed in vivo fundus photographs of the retina and optic nerve head to look for in vivo signs of ocular inflammation. At 1 and 2 weeks these eyes and those of 4 control animals that did not receive the Lac Z constructs were dissected out and stained with X-gal, then processed for cryomicrotomy. In situ RT PCR was perfomed to evaluate transgene expression at the mRNA level. Results - Fundus photography showed no ocular inflammatory response. One week after the CMV-Lac Z injection, the eyecups processed for X-gal showed the blue Lac Z staining of the optic nerve head and retina. Histopathology revealed the blue B-galactosidase reaction product in fibers and glial cells of the optic nerve. Blood vessels in the optic nerve head and peripapillary choroid stained heavily. The X-gal stained retinal ganglion cells and photoreceptor inner segments. Control animals showed no Lac Z staining. By RT PCR the brown mRNA derived reaction product labeled ganglion cells that were also co-labeled by the blue histochemical Lac Z reaction product. Controls with omission of the RT and PCR primers showed only the blue Lac Z staining. Conclusions - AAV viral mediated gene transfer drives (1) measurable tissue expression of foreign proteins and (2) continued transcription of transfected mRNAs in the retina and optic nerves of strain-13 guinea pigs.

Original languageEnglish (US)
Pages (from-to)S640
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

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ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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