Abstract
Purpose :
To characterize lenses in a lens-specific βA3/A1-crystallin conditional Knockout (cKO) mouse model
Methods :
Previously, we have generated a βA3 complete KO mouse model using EUCOM gene trap cassette in exon 4 of mouse CRYAB1 gene (PLOS one, 2016, e0149027). In the present study, the homozygous βA3 KO mice were generated by crossing βA3 flox mice with MLR-10 Cre mice. Heterozygous floxed mice, positive for MLR10 transgene, were backcrossed to βA3fl/fl mice to obtain MLR10 (Cre) hemizygous mice that were homozygous for the foxed allele. These mice (MLR10; βA3fl/fi) are referred as the βA3 cKO mice. Congenital cataract development in the conditional knockout mice was confirmed by Micron-IV Slit-lamp microscopy. Proteins from 1-month-old lenses were analyzed by mass spectrometric and western blot methods.
Results :
As observed previously in the βA3 complete KO mice, lens-specific cKO mice of βA3-crystallin also developed congenital nuclear cataract as confirmed by slit-lamp microscopy. The cKO lenses exhibited age-related increase in the concentration of water-insoluble (WI) proteins and decrease in water-soluble (WS) proteins compared to age-matched wild type (WT) lenses. On separation of lens proteins by low speed (800 g) and high speed (5000 g) centrifugation, the sedimented fractions showed an increased accumulation of protein fragments (< 20 kDa) in cKO lenses compared to WT lenses. Further, MS/MS analysis of these fragments identified a number fragments of different proteins compared to WT lenses. Specifically, an increased accumulation of fragments of β-tubulin, αA-crystallin and phakinin was observed. This was further confirmed by the western blot analysis.
Conclusions :
An absence of βA3-crystallin in the lenses of cKO mice leads to development of congenital cataract, and a greater insolubilization of fragmented lens proteins, which included β-tubulin, αA-crystallin and phakinin.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.