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Hassane Mchaourab, Ping Zou, Wenbiao Chen; Targeted disruption of lens αA-crystallin in Zebrafish (Danio Rerio). Invest. Ophthalmol. Vis. Sci. 2013;54(15):4960. doi: https://doi.org/.
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The chaperone activity of the lens chaperone, α-crystallin, is hypothesized to play a central role in lens transparency by suppressing the aggregation of damaged or modified proteins prone to unfolding. In order to use the zebrafish lens as a model for cataract associated with point mutations in α-crystallin, we sought to assess the role of the endogenous zebrafish α-crystallins in the development of the lens and in its optical properties.
We modified the golden gate TALEN kit and used it to generate two pairs of TALENs targeting the αA-crystallin gene of zebrafish. The synthetic mRNA of the TALEN pairs was injected into zebrafish embryos. Offspring of injected fish were screened for the presence of mutations in targeted region of the αA-crystallin gene.
We found that both TALEN pairs are active in somatic cells by detecting heteroduplex in the PCR product of targeted region from genomic DNA of the embryos 24 hours post mRNA injection. When mature, about 50% of the injected fish transmitted germline mutations in the targeted region to their progeny. Heterozygotes have been identified in 2 independent lines, one with 8 bp deletion and the other with 11 bp deletion. Once the heterozygotes are mature, homozygotes will be generated to determine whether loss of α-crystallin in zebrafish leads to defects in lens development and transparency.
The TALEN method provides an efficient approach to generate knockout of crystallin genes in the zebrafish. A strain null for αA-crystallin would enable an array of experiments to be conducted to assess the contribution of this protein to lens development and function.
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