Extended life of human corneal endothelial cells transfected with the SV40 large T antigen

S. E. Wilson, S. A. Lloyd, Y. G. He, C. S. McCash

Research output: Contribution to journalArticle

70 Scopus citations

Abstract

Purpose. To transfect human corneal endothelial cells with a plasmid vector coding for the SV40 large T antigen to extend the life of the cells in culture. Methods. Human corneal endothelial cells were transfected with the SV40 large T antigen-coding plasmid pSV3neo using the electroporation method. Transfected and control cells were propagated in culture until senescence. Polymerase chain reaction and immunofluorescence were used to demonstrate messenger RNA and protein, respectively, for the Simian virus 40 large T antigen in the transfected cells. Polymerase chain reaction and hot blotting were used to demonstrate messenger RNA coding for several growth factors and receptors in transfected and control cells. Results. The transfected cells continued to proliferate to 38 passages (more than 120 population doublings) in culture (control cells, 8 population doublings). Transfected cells, but not control cells, expressed messenger RNA coding for the Simian virus 40 large T antigen. Similarly, immunofluorescent staining with monoclonal antibodies demonstrated that the Simian virus 40 large T antigen protein was present in the nucleus of the transfected cells. Transfected cells were shown to produce messenger RNA coding for epidermal growth factor, epidermal growth factor receptor, basic fibroblast growth factor, fibroblast growth factor receptor-1, interleukin-1 alpha, the interleukin-1 receptor, transforming growth factor beta-1, and the glucocorticoid receptor. Qualitative expression of the messenger RNA coding for each of these modulators was similar in proliferating primary corneal endothelial cells and proliferating or confluent transfected corneal endothelial cells. Conclusions. In culture, the life of human corneal endothelial cells transfected with a plasmid vector coding for the Simian virus 40 large T antigen is extended. This study suggests that human corneal endothelial cells have the capacity for extensive proliferation, but the proliferation of untransfected cells is regulated through mechanisms that have not yet been characterized.

Original languageEnglish (US)
Pages (from-to)2112-2123
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume34
Issue number6
StatePublished - Jan 1 1993

Keywords

  • Cell culture
  • Cell growth
  • Corneal endothelium
  • Electroporation
  • SV40 large T antigen
  • Senescence
  • Transfection

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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