Development of an efficient method for generation of genetically modified insulin secreting cells and its application for studies on insulin secretion

• Project/Area Number: 15K15351
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Metabolomics
• Research Institution: Nihon University
• Principal Investigator: ISHIHARA Hisamitsu 日本大学, 医学部, 教授 (60361086)
• Co-Investigator(Renkei-kenkyūsha): OTSUKA Yuichiro 日本大学, 医学部, 助手 (40748399)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2015: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: インスリン分泌機構 / 膵β細胞 / 遺伝子発現修飾 / recombinase / インスリン分泌 / リコンビナーゼ / β細胞 / 遺伝子操作

Outline of Final Research Achievements

Genetic modyfication of insulin secreting cells is a powerful strategy to study mechanisms of insulin secretion and of cell death. However, one of the disadvantages encountered in using these cells is the low transfection efficiency of nucleotides. To circumvent the difficulties, we have established a very efficient system based on the recombinase-cassette exchange for generation of a insulin secreting MIN6-derived master cell line.
A recipient platform was generated that has the zeocin-resistant gene flanked with a set of yeast flippase recognition target sites and placed on the genome of MIN6 cells. Overexpression and suppression of gene-of-interest are achieved by delivery with an exchange vector of cDNAs and shRNAs, respectively. Using this cell line, we showed that engineered clones were created within 6 weeks. Moreover, we could generate more than 100 transfectant cell lines and identified novel genes involved in regulation of glucose-stimulated insulin secretion.

Research Products

• [Journal Article] Difficulty diagnosing acute pancreatitis possibly masked by severe hypertriglyceridemia in an obese patient with ketoacidosis-onset type 2 diabetes (2017)
  • Author(s): Fujishiro M, Horita A, Nakagawara H, Mawatari T, Kishigami Y,Tominaga Y, Moriyama M, Ishihara H.
  • Journal Title: Int Med. Volume: 印刷中
  • Peer Reviewed
• [Journal Article] Diabetes mellitus not an unfavorable factor on the prognosis of hepatitis C virus-related hepatocellular carcinoma. (2017)
  • Author(s): Yoshida N, Midorikawa Y, Higaki T, Nakayama H, Tsuji S, Matsuoka S, Ishihara H, Moriyama M, Takayama T.
  • Journal Title: Hepatol Res. Volume: 印刷中 Issue: 1 Pages: 28-35
  • DOI: 10.1111/hepr.12888
  • Peer Reviewed
• [Journal Article] Efficacy and safety of ipragliflozin as add-on therapy to insulin in Japanese patients with type 2 diabetes mellitus (IOLITE): a multi-centre, randomized, placebo-controlled, double-blind study. (2016)
  • Author(s): Ishihara H, Yamaguchi S, Nakao I, Okitsu A, Asahina S.
  • Journal Title: Diabetes Obes Metab. Volume: 18 Issue: 12 Pages: 1207-1216
  • DOI: 10.1111/dom.12745
  • Peer Reviewed
• [Journal Article] Is zinc an intraislet regulator of glucagon secretion? (2016)
  • Author(s): Ishihara H, Wollheim CB.
  • Journal Title: Diabet Int. Volume: 7 Issue: 2 Pages: 106-110
  • DOI: 10.1007/s13340-016-0259-x
  • NAID: 40020883101
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] High-fat diet feeding significantly attenuates anagliptin-induced regeneration of islets of Langerhans in streptozotocin-induced diabetic mice (2015)
  • Author(s): Ｓhinjo T, Nakatsu Y, Iwashita M, Sano T, Sakoda H, Ishihara H, Kushiyama A, Fujishiro M, Nishimura F, Asano T.
  • Journal Title: Diabetol Metab Syndr Volume: ７ Issue: 1 Pages: 50-50
  • DOI: 10.1186/s13098-015-0047-y
  • Peer Reviewed / Open Access
• [Journal Article] DPP-IV inhibitor anagliptin exerts anti-inflammatory effects on macrophages, adipocytes, and mouse livers by suppressing NF-κB activation. (2015)
  • Author(s): Shinjo T, Nakatsu Y, Iwashita M, Sano T, Sakoda H, Ishihara H, Kushiyama A, Fujishiro M, Fukushima T, Tsuchiya Y, Kamata H, Nishimura F, Asano T.
  • Journal Title: Am J Physiol Endocrinol Metab. Volume: 309(3) Issue: 3 Pages: E214-E223
  • DOI: 10.1152/ajpendo.00553.2014
  • Peer Reviewed
• [Journal Article] Octreotide for hypoglycemia caused by sulfonylurea and DPP-4 inhibitor. (2015)
  • Author(s): Yamaguchi, S., Ikejima, M., Furukawa, A., Abe, M., Nakazaki, M., Ishihara, H.
  • Journal Title: Diabetes Res Clin Pract. Volume: 109 Issue: 2 Pages: e8-e10
  • DOI: 10.1016/j.diabres.2015.05.008
  • Peer Reviewed
• [Journal Article] Inhibition ameliorates pancreatic β-cell failure and improves glucose tolerance in the mouse model of Wolfram syndrome (2015)
  • Author(s): Tanji Y, Yamaguchi S, Ishigaki Y, Katagiri H, Oka Y, Ishihara H.
  • Journal Title: J Diabet Mellitus Volume: 5 Issue: 02 Pages: 72-80
  • DOI: 10.4236/jdm.2015.52009
  • Peer Reviewed / Open Access
• [Presentation] Establishment of efficient gene transfer system based on RMCE method in MIN6 cells and new findings of novel genes involved in insulin secretion. (2016)
  • Author(s): Yamaguchi S, Ishihara H.
  • Organizer: 第59回日本糖尿病学会年次集会
  • Place of Presentation: 国立京都国際会館
  • Year and Date: 2016-05-19
  • Invited
• [Presentation] Establishment of efficient gene transfer system based on RMCE method in MIN6 cells, and new findings of novel genes involved in insulin secretion (2016)
  • Author(s): Yamaguchi S, Ishihara H
  • Organizer: 第59回日本糖尿病学会年次学術集会
  • Place of Presentation: 京都国際会議場 ロームシアター みやこめっせ （京都府・京都市）
  • Year and Date: 2016-05-19
  • Invited
• [Presentation] Screening by RMCE-mediated generation of MIN6 cell transformants identifies novel genes important for insulin secretion (2015)
  • Author(s): Furukawa A, Tanaka A, Kosuda M, Otsuka Y, Yamaguchi S, Ishihara H.
  • Organizer: Keystone Symposia Diabetes： New insights into molecular mechanisms and therapeutic strategies.
  • Place of Presentation: ウェスティン都ホテル京都 （京都府・京都市）
  • Year and Date: 2015-10-25
  • Int'l Joint Research
• [Presentation] Screening by RMCE-mediated generation of MIN6 cell transformants identifies novel genes important for insulin secretion (2015)
  • Author(s): Ishihara H, Yamaguchi S
  • Organizer: 第58回日本糖尿病学会年次学術集会
  • Place of Presentation: 海峡メッセ下関 他 （山口県・下関市）
  • Year and Date: 2015-05-21
  • Invited