Abstract
Purpose::
To create a mouse model of human autosomal dominant congenital cataract by knocking-in an alphaA-crystallin mutation in the mouse. A novel missense mutation in CRYAA, the gene encoding alphaA-crystallin, results in a nuclear cataract segregating in a four generation Caucasian family. The C to T transition in exon 1 of codon 49 results in the non-conservative substitution of arginine 49 to cysteine (R49C). We tested the hypothesis that mice carrying this mutation in one allele show early lens opacities, and lenses of mice homozygous for the mutation have a higher degree of lens opacities.
Methods::
Mouse genomic DNA clone derived from a 129Sv strain containing alphaA-crystallin gene was generously provided by Dr. Eric Wawrousek. Knock-in mice were generated by removing the normal gene from one allele by homologous recombination in 129SvJ ES cells to modify the Cryaa gene such that exon 1 contained the R49C mutation, while the second copy of the gene was wild type. ES cells positive for the mutation were 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. Two independent lines of mice, 2005R49CKI3 and 2005R49CKI4, expressing the mutation in Cryaa were bred. Another mouse line, 2005WTKI2 expressing the wild type Cryaa was also generated. 154 heterozygous mice and 43 homozygous mice expressing the mutation were bred and genotyped. To date 28 mice have been examined at 3-5 weeks for cataract formation by slit lamp microscopy, histology and protein biochemistry.
Results::
R49C knock-in mice bred normally and had no lethality associated with the mutation. Nuclear cataract was confirmed in 3/10 mice by slit lamp microscopy. In addition, protein analysis showed a decrease in soluble alpha-crystallin in the heterozygous mice as compared to wild type. Large and small vacuoles were observed in the cortical fibers of 5/10 mice. Vacuoles were more prevalent in homozygous than heterozygous mice. Wild type mice did not show any of these abnormalities.
Conclusions::
This is the first knock-in mouse model for a crystallin mutation associated with hereditary human cataract and establishes the proof of principle that such mice are viable and may be useful for studying the mechanisms of congenital cataract formation.
Keywords: crystallins • chaperones • cataract