Published online Apr 15, 2002. doi: 10.3748/wjg.v8.i2.367
Revised: December 1, 2001
Accepted: December 8, 2001
Published online: April 15, 2002
AIM: Artificial β-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in β-cells is tightly regulated in physiological conditions. The Tet-On system is a “gene switch” system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox.
METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay.
RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0, 10, 100, 200, 400, 800 and 1000 μg•L⁻¹), the level of insulin secreted by the cells treated with Dox (1000 μg·L-1) was 241.0 pU·d-1× cell-1, which was 25-fold that of 9.7 pU·d-1× cell-1 without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 min, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 h following the addition of Dox.
CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial β-cells may be a useful approach to develop cellular therapy for diabetes mellitus.