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Molecular Cloning of the Porcine Insulin cDNA Using a Monolayer Culture of Pancreatic Endocrine Cells

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Porcine pancreatic endocrine cells are an attractive candidate for islet cell transplantation in view of the immunological properties and structural similarities of porcine insulin to human insulin. We recently established a method of isolation and a primary monolayer culture of porcine pancreatic endocrine cells. In this study, cloning of the porcine insulin cDNA was performed to clarify the genetic background of the purified isolated cells. A homology-based PCR cloning method was employed to determine the sequence using mRNA extracted from the monolayer-forming cells, and the candidate products were then determined by a homology search on the human insulin cDNA. According to the newly identified sequence, rapid amplification of cDNA ends was applied to the 5′ and 3′ ends, and the entire cDNA sequence was determined. Gene and protein expression was confirmed by Northern blotting, immunohistochemistry, and enzyme assay. To examine the transcriptional level, the cultured cells were incubated in a 20 mM D-glucose medium in the presence or absence of 5 μM forskolin. The porcine insulin cDNA exhibited a high homology to the human cDNA and showed 85% matching with the human amino acid sequence. D-Glucose at 20 mM stimulated the insulin secretion and mRNA expression, and further addition of 5 μM forskolin with the glucose was applied as the strongest stimulus in this culture system.

Keywords: Key words: Porcine; Molecular cloning; Pancreatic β-cell; RACE (rapid amplification of cDNA ends)

Document Type: Research Article


Affiliations: 1: *Institute of Geriatrics, Aoyama Hospital, Tokyo Women's Medical University, Tokyo, Japan 2: †Department of Medicine IV, Tokyo Women's Medical University, Tokyo, Japan 3: ‡Medical Research Institute, Tokyo Women's Medical University, Tokyo, Japan

Publication date: April 1, 2001

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  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.

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