Establishment of an artificial beta-cell line expressing insulin under the control of doxycycline

World Journal of Gastroenterology : WJG
Xin-Yu QinZe-Guang Han

Abstract

Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-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. 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 regulat...Continue Reading

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Citations

Feb 18, 2004·World Journal of Gastroenterology : WJG·Chen-Xia HeJing-Lun Xue
Dec 27, 2003·World Journal of Gastroenterology : WJG·Yang BaiShou-Yi Yu
Apr 9, 2005·World Journal of Gastroenterology : WJG·Shun LuChun-Yan Zhou
Dec 9, 2009·Journal of Clinical Periodontology·Fernanda O B CorreaSilvana R P Orrico

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