March 1998
Volume 39, Issue 3
Free
Articles  |   March 1998
Retroviral expression of connexins in embryonic chick lens.
Author Affiliations
  • J X Jiang
    Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
  • D A Goodenough
    Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Investigative Ophthalmology & Visual Science March 1998, Vol.39, 537-543. doi:
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      J X Jiang, D A Goodenough; Retroviral expression of connexins in embryonic chick lens.. Invest. Ophthalmol. Vis. Sci. 1998;39(3):537-543.

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

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Abstract

PURPOSE: To develop an in vivo model system in which exogenous proteins can be expressed in embryonic chick lens and to further understand the function of connexin-mediated gap junction intercellular communication in lens cell biology. METHODS: RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells. Retroviral constructs were prepared containing alkaline phosphatase (AP) and FLAG-tagged connexins. Chick lenses were infected in situ by injecting virus into the lumen of lens vesicles at stage 18, cultures were taken at various periods. The lenses were then dissected, and the expressed proteins were visualized by AP histochemical examination and immunostaining. RESULTS: Twenty-four hours after infection, alkaline phosphatase could be seen in epithelia and fibers. As lens fiber maturation progressed, however, the alkaline phosphatase staining was lost as the fibers matured, presumably because of the proteolytic removal of the enzyme. By 72 hours, alkaline phosphatase staining could still be observed in epithelial cells and in differentiating fibers in the bow region but not in the mature lens fibers. FLAG-tagged exogenous lens connexins were also abundantly expressed by viral infection. The exogenous connexins were localized at the cell surfaces in junctional maculae and showed the same cell-type specific distribution as that of their endogenous connexin counterparts. CONCLUSIONS: An in vivo model system has been developed in the chick that provides opportunities to study the expression of wild-type and mutant proteins during lens differentiation. Expression of wild-type connexins has revealed that the characteristic distribution of the three different lens connexins is maintained even when expression is driven by a viral promoter.

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