TALEN-induced knockout of Kif17 in zebrafish

Abstract

Purpose: 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, kif17^sa0119, 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.

Methods: 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.

Results: A line of zebrafish, kif17^mw405, was established containing an 11 base pair deletion early in exon 1 of Kif17 that leads to a premature stop-codon at the 21^st 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.

Conclusions: 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.

Keywords: 689 retina: distal (photoreceptors, horizontal cells, bipolar cells) • 695 retinal degenerations: cell biology • 648 photoreceptors