1. Volume 15, Issue 6, Pages 1195-1202 (June 2007)
Engineered Enteroendocrine Cells Secrete Insulin in Response to Glucose and Reverse Hyperglycemia in Diabetic Mice 
Jaeseok Han, Hyune-Hwan Lee, Hyokjoon Kwon, Seungjin Shin, Ji-Won Yoon, Hee-Sook Jun 
Molecular Therapy 
Volume 15, Issue 6, Pages (June 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Construction of vectors expressing insulin under the control of the GIP promoter. (a) Schematic diagram of the GIP promoter (pGIP) and pGIPi, which contains the GIP promoter region and intron 1. (b) Schematic diagram of pGIPi-ratINS/neo. Amp, ampicillin resistance gene; Neo, neomycin resistance gene; pA, poly A tail; pCMV, cytomegalovirus promoter; ratINS, rat pre-proinsulin complementary DNA (cDNA).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of expression of glucose transporter 2 (GLUT2), glucokinase (GK), and rat insulin messenger RNA (mRNA) in Gi-INS-7 cells. Total RNA was prepared from STC-1 and Gi-INS-7 cells, and the expression of GLUT2, GK, and transgenic rat insulin mRNA (ratINS) was analyzed by RT-PCR. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was analyzed as an internal control. MIN6 cells and intestinal tissue were used as controls, and mouse insulin mRNA (mouseINS) was also analyzed as a control. M, size marker.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Immunocytochemical analysis of glucose transporter 2 (GLUT2), glucokinase (GK), and insulin in Gi-INS-7 cells. (a, b, c) anti-insulin, (d, e, f) anti-GK, or (g, h, i) anti-GLUT2 antibodies were used for staining of (a, d, g) Gi-INS-7, (b, e, h) STC-1, and (c, f, i) HeLa S3 cells. Magnification ×200.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Glucose-responsive insulin secretion in Gi-INS-7 cells in vitro. MIN6 and Gi-INS-7 cells were cultured in the presence of low (5.5 mM) or high (27.5 mM) glucose for 2 hours. (a) Insulin secretion in the culture supernatant and insulin content in cell lysates were measured by enzyme-linked immunosorbent assay. Insulin secretion was calculated as a percentage of the cellular insulin content. (b) Total RNA was isolated from the cells and the expression of insulin mRNA was quantified by real-time reverse transcriptase polymerase chain reaction. Data are expressed as the fold increase in insulin messenger RNA (mRNA) expression over the amount seen in low glucose. Closed bars, MIN6 cells; open bars, Gi-INS-7 cells. ND, insulin mRNA not detectable in STC-1 cells. Values are means ± SEM (n = 6). *P < 0.01, **P <
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Effect of transplanted Gi-INS-7 cells in vivo. Gi-INS-7 or STC-1 cells (5 × 106 cells) were transplanted under the kidney capsule of streptozotocin (STZ)-induced diabetic NOD.scid mice. (a) Blood glucose levels and (b) body weights were measured at the indicated times after transplantation. STZ was injected at day –5. Thirty days after transplantation (arrow), nephrectomy of the graft-bearing kidney capsule was performed. Open circles, Gi-INS-7-transplanted mice (n = 9); open triangles, STZ-induced diabetic mice (n = 4); open squares, STC-1-transplanted mice (n = 4). Values are means ± SEM. Pancreatic sections were prepared from (c) normal, (d) Gi-INS-7-transplanted, and (e) STZ-induced diabetic NOD.scid mice at 3 weeks after transplantation and stained with anti-insulin antibody. Magnification ×40.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Glucose tolerance tests in Gi-INS-7 cell–transplanted NOD.scid mice. (a) Mice went unfed for 6 hours, and glucose (2 g/kg body weight) was administered intraperitoneally. Blood glucose levels were measured at the indicated times following glucose injection; Gi-INS-7-transplanted mice (open circles; n = 3). Non-diabetic NOD.scid mice (open triangles; n = 5) and untreated streptozotocin (STZ)-induced diabetic NOD.scid mice (open squares; n = 3) were used as controls. (b) Serum insulin levels were measured at 0 and 30 minutes after glucose challenge. Closed bars, untreated STZ-induced diabetic mice; hatched bars, non-diabetic mice; open bars, Gi-INS-7-transplanted mice (n = 4 per group). Values are means ± SEM. *P < 0.05; **P < 0.01.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Immunohistochemical analysis of transgenic insulin expression in Gi-INS-7 cells transplanted into streptozotocin (STZ)-induced diabetic NOD.scid mice. (a, b) STC-1 or (c, d) Gi-INS-7 cells (5 × 106 cells) were transplanted under the kidney capsule of STZ-induced diabetic NOD.scid mice. Kidneys containing the transplanted cells were removed when blood glucose levels were normalized, and sections were prepared and stained with anti-insulin antibody (b, d). As a control, sections were stained with only sec-ondary antibody (a, c). Arrows indicate the border between kidney tissue and transplanted cells. Magnification ×100.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions