Tetracycline-regulated secretion of human insulin in transfected primary myoblasts.
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 0.1 microg/ml tetracycline for 48 h inhibited pro/insulin secretion to 19-27% and 7-12%, respectively, compared to tetracycline untreated myoblasts. Following a 48 h tetracycline incubation (1.0 microg/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 72 h. 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.
Scougall
Kathleen T
KT
Diabetes Research Group, School of Clinical Medical Sciences, University of Newcastle upon Tyne, Framlington Place, NE2 4HH, Newcastle upon Tyne, UK.
Shaw
James A M
JA
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D007328:Insulin / Q000235:genetics* / Q000378:metabolism; D000078790:Insulin Secretion; D008297:Male; D018482:Muscle, Skeletal / Q000166:cytology*; D009154:Mutation; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection
D007328:Insulin; D011498:Protein Precursors; D012333:RNA, Messenger; C013737:preproinsulin; D011384:Proinsulin; D013752:Tetracycline