A human beta cell line with drug inducible excision of immortalizing transgenes

Molecular Metabolism
Marion BenazraPhilippe Ravassard

Abstract

Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable fun...Continue Reading

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Citations

Sep 13, 2016·Diabetes, Obesity & Metabolism·R ScharfmannO Albagli
Apr 20, 2017·European Journal of Clinical Nutrition·R CarlessiP Newsholme
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Methods Mentioned

BETA
PCR
transfection
Assay
flow cytometry
ELISA
transgenic

Software Mentioned

Cytogenomics Software
FIJI
FACS Diva
Nexus

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