Purchase this article with an account.
J Samuel Zigler, Eric F Wawrousek, Lixia Lu, Guo-Tong Xu, Debasish Sinha; Cryba1: Do its two gene products (βA3- and βA1-crystallin) have distinct functions?. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5307.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The βA3/A1-crystallin gene (Cryba1) is unique among crystallin genes and unusual for eukaryotic genes in that its mRNA produces 2 proteins through a process called leaky ribosomal scanning or dual translation initiation. Our goal is to determine whether those two proteins, βA3- and βA1-crystallin, have distinct functions.
CRISPR/Cas9 technology was used to modify the sequence of Cryba1 in ways affecting the relative translation of the βA3- and βA1-crystallin proteins. To eliminate expression of the longer βA3-crystallin protein, the first start codon was destroyed by a single nucleotide replacement; to reduce expression of βA1-crystallin without affecting the protein sequence, 5 base pairs were knocked-In before the first start codon to strengthen the Kozak consensus sequence (CCACCATGG), making the first start codon stronger. Mouse lines were generated (Cyagen, Inc) carrying these modified genes and individuals homozygous for the modified genes produced by selective mating. Lenses from these animals were analyzed for protein expression by western blotting and for morphological abnormalities by light microscopy.
Based on densitometric quantification of western blots, wild type mice at 2-4 months of age had about twice as much βA1-crystallin as βA3-crystallin in their lenses. When the Kozak sequence was knocked-in before the first (βA3) start codon in both strands, this ratio was essentially reversed, with βA3 now being the predominant form present. Mice in which the first start codon of Cryba1 had been altered from ATG to GTG in both strands had no detectable βA3-crystallin in their lenses. These mice had a compensatory increase in the level of βA1-crystallin. Lenses from both groups of genetically modified mice were grossly transparent and normal in appearance. The age-matched lens weights were not significantly different in any groups. Histological analyses are in progress.
Further studies are required to determine whether βA3 and βA1 have distinct functions in the lens, although a normal appearing lens is formed in the absence of βA3-crystallin. Further analyses may reveal structural or functional abnormalities in the lenses of the genetically modified mice. It is perhaps more likely that differences will be found in other cell types expressing these proteins, such as retinal pigmented epithelium and ocular astrocytes.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only