A Dual Reporter EndoC-ßH1 Human ß-Cell Line for Efficient Quantification of Calcium Flux and Insulin Secretion.
Endocrinology
; 161(2)2020 02 01.
Article
em En
| MEDLINE
| ID: mdl-31960055
ABSTRACT
Human in vitro model systems of diabetes are critical to both study disease pathophysiology and offer a platform for drug testing. We have generated a set of tools in the human ß-cell line EndoC-ßH1 that allows the efficient and inexpensive characterization of ß-cell physiology and phenotypes driven by disruption of candidate genes. First, we generated a dual reporter line that expresses a preproinsulin-luciferase fusion protein along with GCaMP6s. This reporter line allows the quantification of insulin secretion by measuring luciferase activity and calcium flux, a critical signaling step required for insulin secretion, via fluorescence microscopy. Using these tools, we demonstrate that the generation of the reporter human ß-cell line was highly efficient and validated that luciferase activity could accurately reflect insulin secretion. Second, we used a lentiviral vector carrying the CRISPR-Cas9 system to generate candidate gene disruptions in the reporter line. We also show that we can achieve gene disruption in ~90% of cells using a CRISPR-Cas9 lentiviral system. As a proof of principle, we disrupt the ß-cell master regulator, PDX1, and show that mutant EndoC-ßH1 cells display impaired calcium responses and fail to secrete insulin when stimulated with high glucose. Furthermore, we show that PDX1 mutant EndoC-ßH1 cells exhibit decreased expression of the ß-cell-specific genes MAFA and NKX6.1 and increased GCG expression. The system presented here provides a platform to quickly and easily test ß-cell functionality in wildtype and cells lacking a gene of interest.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Linhagem Celular
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Genes Reporter
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Sinalização do Cálcio
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Células Secretoras de Insulina
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Secreção de Insulina
Tipo de estudo:
Evaluation_studies
/
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article