May 2008
Volume 49, Issue 13
ARVO Annual Meeting Abstract  |   May 2008
A Knockin Mouse Model for the R120G Mutation in AlphaB-Crystallin
Author Affiliations & Notes
  • U. P. Andley
    Deptartment of Ophthalmology, Washington University School of Medicine, St. Louis, Missouri
  • F. Bai
    Dept of Ophthalmology, Washington Univ School of Medicine, St Louis, Missouri
  • J.-H. Xi
    Dept of Ophthalmology, Washington Univ School of Medicine, St Louis, Missouri
  • Footnotes
    Commercial Relationships  U.P. Andley, None; F. Bai, None; J. Xi, None.
  • Footnotes
    Support  NIH R01EY05681, NIH Vision Core grant EY02687 and Research to Prevent Blindness Unrestricted Grant to Department of Ophthalmology at Washington University
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5023. doi:
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      U. P. Andley, F. Bai, J.-H. Xi; A Knockin Mouse Model for the R120G Mutation in AlphaB-Crystallin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5023. doi:

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

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Purpose: : To generate a new model of human autosomal dominant cataract by knocking in an alphaB-crystallin mutation in the mouse. A missense mutation in CRYAB, the gene encoding human alphaB-crystallin, causes desmin-related myopathy and cataracts. The A→G transition in codon 120 of the alphaB-crystallin gene results in the non-conservative substitution of arginine 120 to glycine (R120G). We determined whether knockin mice heterozygous for the R120G mutation in alphaB-crystallin showed changes in lens protein solubility and opacity.

Methods: : Knockin mice were generated by removing one wild type allele by homologous recombination in embryonic stem (ES) cells. Mouse genomic DNA clone from a 129Sv strain containing the alphaB-crystallin gene was generously provided by Dr. Eric Wawrousek. The 5’ and 3’ arms of the Cryab gene were cloned into a plasmid containing a floxed neo cassette. Site-directed mutagenesis was used to modify the Cryab gene such that exon 3 contained the A→G mutation in codon 120. The plasmid was electroporated into SCC10 mouse ES cells. ES cells positive for the mutation were electroporated with Turbo-Cre plasmid to remove the floxed neo cassette, karyotyped, injected into C57BL6 blastocytes and implanted into pseudopregnant ICR females. The chimeric founders were mated with wild type C57BL6 mice and their progeny that genotyped positive for germ line transmission were bred. PCR and Southern blotting were used to verify the mutation. Four independent lines of mice, R120GKI1 to R120GKI4, expressing alphaB-R120G mutation were bred. Fifteen different animals have been examined.

Results: : R120G knock-in mice bred normally and had no lethality associated with the mutation. Three week old alphaB-R120G heterozygous mice developed mild anterior lens opacities. However, biochemical analysis demonstrated an increase in insoluble alphaB-crystallin in the lens fiber cells of heterozygous mice. Co-immunoprecipitation and immunoblot analysis revealed an increase in the interaction of alphaB-crystallin with beta-tubulin in heterozygous lenses, but reduced interaction with gamma-crystallin. Histological analysis showed an increase in vacuoles and disruption of morphology in the anterior cortical fiber cells of heterozygous alphaB-R120G lenses. Wild type lenses did not show any of these changes.

Conclusions: : We have succeeded in creating the first knockin mouse model to recapitulate the human R120G mutation in alphaB-crystallin. Our results demonstrate that knockin lenses have increased protein insolubility and display morphological defects at eye opening as compared with wild type lenses.

Keywords: crystallins • mutations • chaperones 

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