Using recombinase-mediated cassette exchange to engineer MIN6 insulin-secreting cells based on a newly identified safe harbor locus

Asami Furukawa, Aya Tanaka, Suguru Yamaguchi, Minami Kosuda, Midori Yamana, Akiko Nagasawa, Genta Kohno, Hisamitsu Ishihara

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

Aims/Introduction: Recent studies have identified genomic and transcript level changes along with alterations in insulin secretion in patients with diabetes and in rodent models of diabetes. It is important to establish an efficient system for testing functional consequences of these changes. We aimed to generate such a system using insulin-secreting MIN6 cells. Materials and Methods: MIN6 cells were first engineered to have a tetracycline-regulated expression system. Then, we used the recombination-mediated cassette exchange strategy to explore the silencing-resistant site in the genome and generated a master cell line based on this site. Results: We identified a site 10.5 kbps upstream from the Zxdb gene as a locus that allows homogenous transgene expression from a tetracycline responsible promoter. Placing the Flip/Frt-based platform on this locus using CRISPR/Cas9 technology generated modified MIN6 cells applicable to achieving cassette exchange on the genome. Using this cell line, we generated MIN6 subclones with over- or underexpression of glucokinase. By analyzing a mixed population of these cells, we obtained an initial estimate of effects on insulin secretion within 6 weeks. Furthermore, we generated six MIN6 cell sublines simultaneously harboring genes of inducible overexpression with unknown functions in insulin secretion, and found that Cited4 and Arhgef3 overexpressions increased and decreased insulin secretion, respectively. Conclusions: We engineered MIN6 cells, which can serve as a powerful tool for testing genetic alterations associated with diabetes, and studied the molecular mechanisms of insulin secretion.

Original languageEnglish
Pages (from-to)2129-2140
Number of pages12
JournalJournal of Diabetes Investigation
Volume12
Issue number12
DOIs
Publication statusPublished - Dec 2021

Keywords

  • Insulin secretion
  • Recombination-mediated cassette exchange
  • Tetracycline-regulated expression

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