July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Monitoring the developing photoreceptors in the hiPSC-derived three dimension retinal organoid culture using bicistronic 2A-peptide-based co-expression reporter knock-in system
Author Affiliations & Notes
  • Kohei Homma
    Ophthalmology, Keio University School of Medicine, Tokyo, Japan
  • Nami Ozato
    Ophthalmology, Keio University School of Medicine, Tokyo, Japan
  • Kazuo Tsubota
    Ophthalmology, Keio University School of Medicine, Tokyo, Japan
  • Hideyuki Okano
    Physiology, Keio University School of Medicine, Tokyo, Japan
  • Yoko Ozawa
    Ophthalmology, Keio University School of Medicine, Tokyo, Japan
  • Footnotes
    Commercial Relationships   Kohei Homma, Wakasa Seikatsu (F); Nami Ozato, None; Kazuo Tsubota, None; Hideyuki Okano, None; Yoko Ozawa, Wakasa Seikatsu (F)
  • Footnotes
    Support  Grant-in-Aid for Scientific Research (C) (16K11305) from Japan Society for the Promotion of Science (JSPS).
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 1983. doi:
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      Kohei Homma, Nami Ozato, Kazuo Tsubota, Hideyuki Okano, Yoko Ozawa; Monitoring the developing photoreceptors in the hiPSC-derived three dimension retinal organoid culture using bicistronic 2A-peptide-based co-expression reporter knock-in system. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1983.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To label the specific cell lineages, knock-in technology is often used in human pluripotent stem cells. However, in conventional knock-in strategy, the target gene is replaced by knock-in reporter and one of the target alleles is deleted. This study tested the bicistronic 2A-peptide-based co-expression (B2AC) approach to label the target gene without deleting one allele.

Methods : Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) and gRNA DNA vectors were used to induce DNA double strand break around the stop codon of the target genes, those were a pan-photoreceptor marker, Crx, and a cone photoreceptor marker, Pde6H, in the human induced pluripotent stem cells (hiPSCs: 454E2, RIKEN). The 2A peptide sequence, the fluorescent protein gene, and the antibiotic resistant gene expression cassette were inserted at 3’-end of the target genes by the homologous recombination with donor vectors. The established Crx::2A::E2-Crimson and Pde6H::2A::YFP double-knock-in hiPSCs were differentiated into retinal organoids by three dimension retinal differentiation culture.

Results : During differentiation, each target gene expression and fluorescent intensity in the cells were positively correlated, suggesting that the B2AC-mediated reporters represented the target gene expression. The immunohistochemistry of target proteins and the maturation of fluorescent reporter cells after long-term differentiation culture indicated that knock-in of the reporter gene did not affect the function of the target genes and retinal organoid development. By fluorescence activated cell sorting (FACS), the reporter positive cells were purified and analyzed their gene expression profiles. B2AC reporter successfully represented Crx and Pde6H upregulation during retinal differentiation from hiPSC.

Conclusions : These results validated the B2AC reporter knock-in approach enabled to monitor the expression of target genes during photoreceptor development in the iPS-derived three dimension retinal organoid. This approach may be utilized for analyzing retinal development, disease signaling, and drug screening, as well as cell transplantation.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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