April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
TALEN-induced knockout of Kif17 in zebrafish
Author Affiliations & Notes
  • Tylor Lewis
    Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI
  • Jason Bader
    Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI
  • Peter Volberding
    Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI
  • Jonathan Bostrom
    Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI
  • Ross F Collery
    Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI
  • Brian A Link
    Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI
  • Joseph C Besharse
    Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI
  • Footnotes
    Commercial Relationships Tylor Lewis, None; Jason Bader, None; Peter Volberding, None; Jonathan Bostrom, None; Ross Collery, None; Brian Link, None; Joseph Besharse, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 382. doi:https://doi.org/
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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)

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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, 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.

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, 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.

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  
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