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Tetracycline-regulated secretion of human insulin in transfected primary myoblasts

Lookup NU author(s): Dr Kathleen Scougall, Professor James ShawORCiD

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Abstract

A mechanism for safely regulating transgene expression will be necessary for gene therapy approaches to endocrine disorders. In this study, a two-plasmid tetracycline-inducible system was used to regulate expression of human proinsulin (hppI1) and a mutated proinsulin construct (hppI4, allowing cleavage by furin) in primary rat soleus myoblasts. In hppI1 and hppI4 transient transfections, the presence of 0.01 and 0.1μg/ml tetracycline for 48h inhibited pro/insulin secretion to 19-27% and 7-12%, respectively, compared to tetracycline untreated myoblasts. Following a 48h tetracycline incubation (1.0μg/ml), pro/insulin secretion in hppI1 and hppI4 transfected myoblasts was reduced to <4% of that in cells incubated without tetracycline. Pro/insulin secretion equivalent to that of untreated cells was restored following tetracycline withdrawal and incubation for a further 72h. Conversion of proinsulin to insulin in transfected myoblasts was <1% for hppI1 and >45% for hppI4. In conclusion, regulated insulin secretion has been achieved in a dose-dependent and reversible manner in primary myoblasts. © 2003 Elsevier Science (USA). All rights reserved.

Publication metadata

Author(s): Scougall KT, Shaw JAM

Publication type: Article

Publication status: Published

Journal: Biochemical and Biophysical Research Communications

Year: 2003

Volume: 304

Issue: 1

Pages: 167-175

ISSN (print): 0006-291X

ISSN (electronic): 1090-2104

Publisher: Academic Press

URL: http://dx.doi.org/10.1016/S0006-291X(03)00553-9

DOI: 10.1016/S0006-291X(03)00553-9

PubMed id: 12705902

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