Transfection of luciferase gene by gold particle bombardment in rabbit organ cultured corneas

J. C. Tsai, J. S. Whitsitt, J. M. Davidson, M. S. Chang, D. M. O'Day, D. J. Shetlar, R. D. Robinson

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Purpose. The use of particle bombardment for gene transfer has been successfully reported in dermal tissue. This study was undertaken to determine the feasibility of gene transfer of luciferase cDNA by gold particle bombardment in organ cultured corneas. Methods. Corneas from New Zealand white albino rabbits were placed in a organ culture medium for 5 days prior to gene transfer. A cytomegalovirus promoter-luciferase construct derived from a pGL2 vector was used as a reporter gene. The Accell® Pulse Gun (gene gun) delivery device (Agracetus, Middleton, WI) was used for particle bombardment. A suspension of expression plasmid along with gold particles (0.95μ in diameter) was dried in the interior of Tefzel tubing (1/8″ b.d.). The tubing was cut 1/2″ in length; each ha inch segment contained approximately 0.5 mg of DNA-coated gold particles. The 1/2 tubes were loaded into the gene gun, and the muzzle of the gene gun was positioned 2 cm over the organ cultured corneas. The DNA coated gold particles were accelerated into the corneas by blasts of helium ranging in pressure from 200 to 300 psi. The corneas were transferred into fresh culture medium. After 24 hrs, luciferase activity was assayed with the Luciferase Assay System® (Promega) and standardized to the amount of protein. Histological studies were also performed. Results. The transfected corneas demonstrated luciferase activity of 244-890 units/μg of protein. The control corneas demonstrated < 20 units/μg. Histological sections revealed the gold particles to be embedded in the cytoplasm of the epithelial cells. At 200 psi, the gold particles were predominately observed in the outer epithelial cells. At 300 psi, the gold particles were distributed throughout the corneal epithelial cell layer. Conclusions. Gene transfer in corneal tissue can be accomplished by gold particle bombardment and may be a potential delivery system for ocular gene therapy.

Original languageEnglish
Pages (from-to)S683
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - 15 Feb 1996
Externally publishedYes

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