Transgenic Expression of Cx50 Leads to Cataract Formation

Abstract

Abstract: : Purpose: To study the effects of altered connexin abundance and isotype ratio on mouse lens structure and function by transgenic expression of human Cx50 (hCx50). Methods: Transgenic mice were generated using a DNA construct containing the hCx50 coding region and a C–terminal FLAG epitope driven by the chicken ßB1–crystallin promoter. Insertion of the transgene into genomic DNA was demonstrated by PCR. Protein levels and distribution were studied by immunoblotting and immunofluorescence. Lens structure was studied by light microscopy and by transmission (TEM) and scanning (SEM) electron microscopy. Lens transparency and function were assessed by visual inspection and laser scanning. Results: Three lines of transgenic mice had visible cataracts and expressed the transgenic protein. Cx50 was detected as a single band by immunoblotting. Transgenic lenses were smaller than lenses from non–transgenic littermates. Most lenses from transgenic animals had severe opacities that precluded quantitative analysis of optical quality. In the few instances in which laser scanning could be performed, the optical quality was significantly compromised. Light microscopy showed areas of liquefaction, a displaced bow region and persistent fiber cell nuclei. SEM showed altered fiber structure and fiber–fiber interactions. TEM showed intracellular vesicles. Anti–Cx50 immunoreactivity was detected at appositional membranes in all lenses, but transgenic lenses also showed localized staining in the cytoplasm. No significant changes in aquaporin 0 or N–cadherin distribution were evident; however, both revealed cell shape changes in the transgenic animals. No significant changes in abundance or solubility of αA–, αB– or ß–crystallins were observed. No difference in Cx50 solubility in RIPA buffer was observed between non transgenic and transgenic animals. Conclusions: These results demonstrate that transgenic expression of Cx50 leads to cataracts associated with several aberrations in lens fiber structure and incomplete or slowed cell differentiation.

Keywords: gap junctions/coupling • cell adhesions/cell junctions • transgenics/knock-outs