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

Autor: Aya Tanaka, Midori Yamana, Hisamitsu Ishihara, Suguru Yamaguchi, Akiko Nagasawa, Genta Kohno, Asami Furukawa, Minami Kosuda
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Zdroj: Journal of Diabetes Investigation, Vol 12, Iss 12, Pp 2129-2140 (2021)
Journal of Diabetes Investigation
ISSN: 2040-1116
2040-1124
Popis: 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.
A platform for recombinase‐mediated cassette exchange is placed by a genetic editing method. This allows easy and efficient generation of insulin‐secreting cells with modified expressions of genes of interest.
Databáze: OpenAIRE
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