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Tylor Lewis, Jason Bader, Peter Volberding, Jonathan Bostrom, Ross F Collery, Brian A Link, Joseph C Besharse; TALEN-induced knockout of Kif17 in zebrafish. Invest. Ophthalmol. Vis. Sci. 2014;55(13):382. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previous work using both dominant-negative Kif17 and morpholino-mediated knockdown of Kif17 has shown a crucial role for the kinesin-II family member in the initial development of photoreceptor OS. However, these methods limited the analysis to the initial 5 dpf. To further establish the function of Kif17 within photoreceptors, permanent knockout is required. A line, kif17sa0119, that was reportedly null for Kif17 was found to have delayed peripheral OS morphogenesis at 3 dpf but recovered by 5 dpf. However, the "null" mutant was found to be hypomorphic with only 50% reduction in Kif17 levels. We, therefore, sought to produce a true knockout allele by gene targeting.
We used transcription activator-like effector nucleases (TALENs), a recent technology in genetic editing, to target Kif17 exon 1 to generate a double-stranded break. Repair by the error-prone non-homologous end joining was expected to create frame-shift mutations leading to a functional Kif17 knockout. Mutations were analyzed through genomic PCR and sequencing and mRNA expression was analyzed by qPCR. Retinas were then analyzed morphologically using a combination of histology and optical coherence tomography (OCT), an emerging technique that allows for the high-resolution imaging of a retina through measuring backscattered or reflected light both in real time and in vivo.
A line of zebrafish, kif17mw405, was established containing an 11 base pair deletion early in exon 1 of Kif17 that leads to a premature stop-codon at the 21st codon, generating a functional Kif17 knockout. Contrary to previous work, Kif17 knockouts exhibited normal OS formation within the first 6 days of development. However, there is progressive disorganization of the outer retina, particularly observed as the loss of the ordered tiering of cone OS at 10 weeks.
Kif17 knockout does not appear to significantly affect OS morphogenesis as previously suggested. However, there appears to be a long-term, progressive outer retinal disorganization phenotype that could not be studied with the previous transient methods. With an established stable line of Kif17 knockout fish, long-term evaluation of the progression of disorganization, as well as investigation of the underlying mechanisms, is possible.
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