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Derek R. Rhodes, Leonel Trujillo, Isabel Rushanaedy, Chris Boniface, Kirsten Lampi; Importance of βB1 Crystallin in Development of Zebrafish. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2772.
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© ARVO (1962-2015); The Authors (2016-present)
The βB1-crystallin is a major protein in the young lens, including in zebrafish. We propose that βB1 serves a major role in lens formation and function. βB1 has several isoforms & expression of the main form is high on day 4 related to other proteins (Greiling, Molecular Vision 2009).
Expression of βB1 in zebrafish was knocked-down in order to test its role in lens development. Adult AB strain fish were purchased from ZIRC(Zebrafish International Resource Center_Eugene, OR) and bred in order to microinject into embryos. Antisense DNA morpholinos(MOs) were designed based on the target βB1 main DNA sequence and were injected into the yolk of the embryo during the 1-2 cell stage of development. Embryos were harvested at 24, 48 and 72hpf (hours post fertilization) and then de-chorinated, photographed, and decapitated prior to being homogenized for RNA extraction. After the RNA was quantified, cDNA synthesis was done using an iScipt kit. Relative levels of βB1 between knockdown and control groups were done via qPCR standard curve analysis. Protein levels were estimated via SDS. The experiment was repeated.
At 48hpf the averaged results show a mortality rate of 11.6% for the uninjected, 11.9% for the control MO, and 14.3% for the test MO groups. No toxicity affects were found in the uninjected and control groups, however, 9.6% of the test MO group showed signs of non-specific MO toxicity. A phenotype of a mal formed lens was observed 88% of the time in the test MO group, but not in the uninjected or control MO groups. Test MO groups were found to have 21% βB1 compared to the uninjected group via qPCR analysis. One and two dimension gel analysis indicates a difference in overall protein expression; this is being further explored.
Knocking down βB1 during development leads to phenotypic changes at 48hpf supporting the role that βB1 is needed for lens development. Given the high levels of expression of βB1-main at 4 days, it appears that the protein is significantly important and the other crystallins cannot compensate for the loss. In the future we will be knocking down the expression of this gene and performing a rescue with a mutant deaminated form of the protein that will greatly increase our understanding of the role of βB1 related to lens development and cataract formation.
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