Abstract
Purpose: :
SPARC is normally and predominantly expressed in lens epithelium, and is significantly decreased in differentiated fiber cells. SPARC–null mice develop cataracts, characterized by alterations in the lens capsule and by vacuole formation within the lens fibers at the equator. To understand the role of SPARC in cataractogenesis, we generated transgenic mice overexpressing SPARC in lens fiber cells under the control of the lens αA–crystallin promoter.
Methods: :
Full–length murine (m)SPARC cDNA was ligated into the vector pCPV6 containing the mouse αA–crystallin promoter. The mSPARC transgenic cDNA was microinjected into pronuclear stage B6C3 embryos. Founder mice were identified by Southern blot and PCR, and were subsequently mated to wt C57BL/6 mice. Histology, immunohistochemistry and immunoblotting were performed on transgenic lenses at various ages.
Results: :
The first pronuclear microinjection resulted in 6 newborn mice, of which one was transgenic. One transgenic line was established with the expression of SPARC protein in lens epithelium and lens fiber cells, by immunohistochemistry and immunoblotting with antibodies against mouse SPARC. Mice homozygous for expression of the transgene showed SPARC protein in primary fibers, and abundant SPARC in secondary lens fibers; in hemizygous transgenic mice, SPARC was predominantly detected in lens fibers at the periphery cortex. Light microscopy of H&E–stained lens sections from homozygous mice revealed vacuoles in the swollen fiber cells in cortex 5 days after birth. These changes were not observed in hemizygous mice of the same age. Transgenic lenses have a normal size and a grossly normal shape compared to their non–transgenic counterparts at postnatal ages examined. Lens opacity was not observed in young (1 month) transgenic lenses.
Conclusions: :
Mice hemizygous for SPARC transgene appear to have clear lenses with normal morphology. Overexpression of SPARC in lenses of homozygous transgenic mice affected secondary lens fiber cell morphology. SPARC appears to be an important factor for lens development and fiber cell differentiation.
Keywords: transgenics/knock-outs • gene/expression • cataract