June 1999
Volume 40, Issue 7
Free
Articles  |   June 1999
Exogenous gene expression and protein targeting in lens fiber cells.
Author Affiliations
  • V I Shestopalov
    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
  • S Bassnett
    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Investigative Ophthalmology & Visual Science June 1999, Vol.40, 1435-1443. doi:
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      V I Shestopalov, S Bassnett; Exogenous gene expression and protein targeting in lens fiber cells.. Invest. Ophthalmol. Vis. Sci. 1999;40(7):1435-1443.

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

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Abstract

PURPOSE: To test the ability of lens fiber cells at various stages of differentiation to transcribe and translate microinjected DNA templates. METHODS: Expression plasmids encoding green fluorescent protein (GFP) or a GFP-tagged membrane protein (human CD46) were microinjected into organ-cultured embryonic chicken lenses. Protein expression was visualized by confocal microscopy. RESULTS: GFP expression was detected within 12 hours of microinjection, evenly distributed throughout the cytoplasm of the injected cell. All nucleated fiber cells were competent to express GFP, whereas the anucleated central fiber cells were not. When GFP was fused to the C-terminal of CD46, the fusion protein was synthesized intact and properly inserted in the fiber cell plasma membrane. In contrast, N-terminal fusions were cleaved during synthesis, resulting in retention of the GFP tag in the endoplasmic reticulum. CONCLUSIONS: Microinjection of expression plasmids is an effective technique for introducing exogenous genes into individual fiber cells. With this approach, the results show that fiber cells are transcriptionally and translationally competent until the time of organelle loss, and that fiber cells deep within the lens are capable of synthesizing new plasma membrane proteins. The techniques described here should have broad application in studies of fiber cell differentiation and provide a useful complement to conventional transgenic approaches.

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