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Control of Autoimmune Diabetes in NOD Mice by GAD Expression or Suppression in B Cells

Control of Autoimmune Diabetes in NOD Mice by GAD Expression or Suppression in B Cells

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Control of Autoimmune Diabetes in NOD Mice by GAD Expression or Suppression in B Cells

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  1. Control of Autoimmune Diabetes in NOD Mice by GAD Expression or Suppression in B Cells Yoon, J. W., C. S. Yoon, H.W. Lim, Q. Q. Huang, Y, Kang, K. H. Pyun, K. Hirasawa, R.S. Sherwin, and H. S. Jun. 1999. Science 284: 1183-1187. Empress Hughes Dr. Peter Lin 10 March 2003

  2. Type 1 Diabetes Mellitus • Insulin-dependant • Destruction of the B-cells in the islet of the pancreas due to T-cell mediated auto-immune destruction of the cells • which causes a decrease in insulin

  3. Glutamic Acid Decarboxylase • Found in humans and non-obese mice (NOD) • Thought to be the auto antigen responsible for triggering B-cell specific autoimmunity (no hard evidence yet) • Provokes early T cell proliferation in NOD mice • Two isoforms: • 65kb • 67kb

  4. TERMS • GAD- Glutamic Acid Decarboxylase, the auto-antigen looked at in this study • Type I Diabetes- characterized by beta cells not beig able to produce insulin • RIP- Rat insulin promoter • Immunohistochemistry- a stain that enables the viewer to visualize the changes in a sample (ie. Levels of GAD expression)

  5. Purpose • To determine the effects of GAD expression on the development of autoimmune diabetes melitus type 1 in NOD mice.

  6. DNA RNA PROTEIN What is Antisense? Central Dogma

  7. Methods Used • Transgene created (A). • Southern blot and PCR was used to confirm the presence of the transgene (B). • Antisense expression (C). • Northern blot showing antisense GAD expression (D). • GAD suppression analyzed using a western blot (E). • Levels of GAD expression was tested sing Immunohistochemistry (F). *Each will be explained in more detail during the presentation

  8. Transgenic mice with with an antisense GAD transgene suppressed beta cell GAD expression Fig. Showing the suppression of GAD • RIP was used as a control • rGAD65 and rGAD67 are isoforms of rat cDNA for the antisense strand • When the antisense was made by rat cDNA, GAD was NOTexpressed

  9. Expression of antisense GAD transcript by reverse transcriptase PCR • There were six lanes of antisense GAD65 and GAD67 determined by level of expression • #57 had a high antisense GAD mouse • The offspring of this generation expressed GAD at very high levels • )

  10. Classification of Expression The lines were classified as the following: High (H-AS-GAD-NOD), Medium (HA-AS-GAD-NOD) Low (HA-AS-GAD-NOD High (Hk-AS-GAD-NOD) Medium (Mk-AS-GAD-NOD) Low (Lk-AS-GAD-NOD) First Three Lines Second Three Lines

  11. Immunohistochemisty • Stain that shows level of expression • Level of expression was quantities by introducing anti-bodies to GAD and insulin • Many levels of GAD expression was found in the transgene negative mouse

  12. Who had the most expression? • L-AS-GAD-NOD showed slight suppression of GAD • M-AS-GAD-NOD showed some suppression of GAD • While H-AS-GAD-NOD showed the greatest amount (total) of suppression of GAD

  13. Discussion • GAD expression was found to be necessary for the development of diabetes in NOD mice • Anti-sense gene not a major contributor to the prevention of diabetes