Targeted genomic deletion of the lens-specific intermediate filament protein CP49.
OBJECTIVE
To deduce the function of the lens-specific cytoskeletal structure, the beaded filament, by blocking expression of the fiber cell-specific beaded filament protein CP49.


METHODS
The first exon of the mouse CP49 gene was deleted by using targeted genomic deletion techniques. Gene deletion was assessed through Southern blot analysis and PCR. Translation and protein expression were characterized by Northern and Western blot analysis of both CP49 and its assembly partner filensin. The architecture of knockout lenses was compared with that of wild-type lenses at the histologic level by light microscopy. Lens clarity was assessed in situ by direct ophthalmic examination and slit lamp microscopy.


RESULTS
Transcription and translation of CP49 were successfully negated in knockout animals. Lenses homozygous for the CP49 deletion showed no obvious changes in lens architecture at the light microscope level. Filensin levels were sharply reduced, although filensin mRNA levels appeared unchanged. Direct examination of lenses showed no obvious loss of lens clarity, but slit lamp examination revealed the emergence of opacification in even the youngest animals. The opacification worsened with age.


CONCLUSIONS
The absence of CP49 causes a subtle loss of optical clarity in the ocular lens, a loss that worsens with age. However, CP49 is not essential for the assumption or maintenance of overall fiber cell shape or long-range order of fiber cells. CP49 appears to regulate the protein levels of its assembly partner filensin, suggesting a mechanism for the regulation of beaded filament protein stoichiometry.
Alizadeh
Azita
A
Department of Cell Biology and Human Anatomy, University of California, Davis 95616, USA.
Clark
John I
JI
Seeberger
Teri
T
Hess
John
J
Blankenship
Tom
T
Spicer
Andrew
A
FitzGerald
Paul G
PG
D000375:Aging; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002386:Cataract / Q000209:etiology / Q000378:metabolism* / Q000503:physiopathology; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017353:Gene Deletion; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007908:Lens, Crystalline / Q000378:metabolism* / Q000503:physiopathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism
D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D012333:RNA, Messenger; C079098:filensin; C084808:phakinin