Characterization of a mutation in the lens-specific CP49 in the 129 strain of mouse.

Purpose: The 129 strain of mouse carries a mutation in the gene for CP49 (phakinin), an intermediate filament protein thus far demonstrated only in the lens fiber cell. As such, these mice represent naturally occurring mutants of interest in the study of the lens cytoskeleton. However, this strain of mouse is also widely used as a source of embryonic stem cells in gene-targeting studies. The presence of a mutation in a lens-specific gene can confound interpretation of studies in which lens genes have been knocked out. In the present study, both the genotype and phenotype of these mice were characterized, to permit an evaluation of the biological impact of this mutation and to facilitate the discrimination between wild-type and mutant animals that have been derived from this strain in gene-targeting studies.

Methods: The CP49 cDNA and, when relevant, the genomic DNA sequences were determined for the 129/SvJ and C57BL/6J mice and from a commercially available 129/OLa P1 genomic clone. PCR primers were screened for their capacity to discriminate between the mutant and wild-type CP49 genes. Northern blot analysis was used to assess mRNA levels for CP49, filensin, and gammaS-crystallin (control). Western blot analysis was used to identify changes in protein size and abundance. The impact of the mutation on lens architecture was evaluated at the light-microscope level. Lens fiber cell ghosts from mutant and wild-type mice were examined in the electron microscope for the presence of beaded filaments. Lens clarity was assessed by slit lamp.

Results: The 129 strain of mice exhibited a 6303-bp deletion from the end of intron B, and the beginning of exon 2. This deletion results in the loss of the exon 2 splice acceptor site, absence of exon 2 from the CP49 mRNA, and dramatically reduced levels of CP49 mRNA. The CP49 protein was undetectable by Western blot analysis. Messenger RNA levels for filensin, CP49's assembly partner, were normal, but protein levels were sharply reduced. Light microscopy established that the initial differentiation and elongation of the fiber cells proceeded normally. Electron microscopy showed the absence of beaded filaments, whereas slit lamp microscopy showed a slowly emerging and progressive loss of optical clarity.

Conclusions: The 129/SvJ and 129/OLa strains of mice harbor a mutation that sharply reduces CP49 mRNA levels and essentially eliminates both CP49 and the beaded filament. These lenses exhibited a slow but progressive loss of optical clarity with age. Thus, the 129 strain of mouse behaves as a functional CP49 knockout. The loss of clarity in the lenses of these animals and the absence of beaded filaments (and any attendant interactions that may exist between beaded filaments and other lens proteins/structures) suggest that gene-targeting studies of lens proteins in which the 129 strain was used as a source of embryonic stem cells may need reevaluation.