Physiological and morphological characterization of trangenic pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPR_1hnd_1hnn) [Elektronische Ressource] : a large animal model of diabetes research / von Christiane Fehlings

Christiane Fehlings

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 28
• Poids de l'ouvrage: 8 Mo

Extrait

Aus dem
Lehrstuhl für Molekulare Tierzucht und Biotechnologie
(Prof. Dr. E. Wolf)
und dem
Lehrstuhl für Tierpathologie
(Prof. Dr. W. Hermanns)
der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München


Arbeit angefertigt unter der Leitung von
Prof. Dr. E. Wolf und Prof. Dr. R. Wanke


Physiological and morphological characterization of transgenic pigs
expressing a dominant-negative glucose-dependent insulinotropic polypeptide
dn
receptor (GIPR ) – a large animal model for diabetes research


Inaugural Dissertation
zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München


von
Christiane Fehlings
aus Günzburg


München 2010 Gedruckt mit der Genehmigung der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München











Dekan: Univ.-Prof. Dr. J. Braun

Berichterstatter: Univ.-Prof. Dr. E. Wolf

Korreferent/en: Univ.-Prof. Dr. Wanke
Univ.-Prof. Dr. Kaspers
Univ.-Prof. Dr. Hartmann
Univ.-Prof. Dr. Potschka









Tag der Promotion:
24. Juli 2010









Meinen Eltern

During the preparation of this work the following paper has been published:

Renner, S., Fehlings, C., Herbach, N., Hofmann, A., von Waldthausen, DC.,
Keßler, B., Ulrichs, K., Chodnevskaja, I., Moskalenko, V., Amselgruber, W.,
Goeke, B., Pfeifer, A., Wanke, R. and Wolf, E. (2010) ”Glucose intolerance and
reduced proliferation of pancreatic -cells in transgenic pigs with impaired GIP
function.” Diabetes (published online ahead of print 2010/02/25; doi: 10.2337/db
09-0515).

Table of contents
1 Introduction..................................................................1
2 Review of the literature................................................3
2.1 The incretin hormone system .................................................... 3
2.2 Glucose-dependent insulinotropic polypeptide (GIP) ............. 4
2.2.1 Synthesis, secretion and degradation of GIP................................ 4
2.2.2 GIP receptor and signal transduction............................................ 4
2.2.3 Biological actions of GIP ............................................................... 5
2.2.3.1 The endocrine pancreas ............................................................... 6
2.2.3.2 Pro-proliferative and anti-apoptotic effects of GIP on -cells ........ 7
2.2.3.3 Adipose tissue............................................................................... 9
2.2.3.4 Bone.............................................................................................. 9
2.2.3.5 Nervous system .......................................................................... 10
2.3 Glucagon-like peptide-1 (GLP-1).............................................. 10
2.3.1 Secretion, synthesis and degradation ......................................... 10
2.3.2 GLP-1 receptor and signal transduction...................................... 11
2.3.3 Biological actions of GLP-1......................................................... 11
2.4 The contribution of GIP and GLP-1 to type 2 diabetes
mellitus ...................................................................................... 12
2.5 Incretins in diabetes research ................................................. 13
-/-
2.5.1 GIP receptor knockout mice (GIPR ) ......................................... 13
-/-
2.5.2 GLP-1 receptor knockout mice (GLP-1R ) ................................. 16
2.5.3 Double incretin receptor knockout mice (DIRKO) ....................... 17
2.5.4 Mice expressing a dominant-negative GIPR
dn
(GIPR transgenic mice) ............................................................ 18
2.5.5 GIP transgenic mice.................................................................... 19
2.5.6 Prolonging the action of GIP, GLP-1 or both............................... 19
2.6 The pig as an animal model in research ................................. 21
Table of contents II
2.6.1 Genetically modified pigs for translational research.................... 22
2.6.1.1 Cardiovascular disease............................................................... 23
2.6.1.2 Cerebral diseases ....................................................................... 23
2.6.1.3 Ophthalmic disease..................................................................... 24
2.6.1.4 Motor neuron disease ................................................................. 24
2.6.1.5 Cystic fibrosis.............................................................................. 24
2.6.1.6 Diabetes...................................................................................... 25
2.6.2 Pigs as models in type 2 diabetes mellitus research................... 25
2.6.2.1 Yucatan Minipigs......................................................................... 26
2.6.2.2 Sinclair minipigs .......................................................................... 27
2.6.2.3 Göttingen minipigs ...................................................................... 27
2.6.2.4 Yorkshire strains ......................................................................... 29
2.6.2.5 Chinese Guizhou minipig ............................................................ 30
dn
2.7 GIPR transgenic pigs............................................................. 30
dn
2.7.1 Generation of GIPR transgenic pigs......................................... 31
2.7.2 Physiological characterization..................................................... 32
2.7.3 Morphological characterization ................................................... 36
3 Animals, Materials and Methods...............................39
3.1 Pigs ............................................................................................ 39
3.2 Materials .................................................................................... 39
3.2.1 Apparatuses................................................................................ 39
3.2.2 Consumables .............................................................................. 40
3.2.3 Chemicals ................................................................................... 41
3.2.4 Antibodies, drugs, enzymes and other reagents......................... 43
3.2.4.1 Antibodies ................................................................................... 43
3.2.4.1.1 Primary antibodies ...................................................................... 43
3.2.4.1.2 Secondary antibodies.................................................................. 43
3.2.4.2 Drugs .......................................................................................... 44
3.2.4.3 Enzymes ..................................................................................... 44
3.2.4.4 Other reagents ............................................................................ 44 Table of contents III
3.2.5 Buffers, media and solutions....................................................... 45
3.2.5.1 Chloroform-isoamylalcohol (CiA) ................................................ 45
3.2.5.2 Citrate buffer (pH 6.0) ................................................................. 45
3.2.5.3 Citrate buffer for cleaved caspase-3 IHC (pH 6.0) ...................... 45
3.2.5.4 Phenol-chloroform-isoamylalcohol (PCiA)................................... 45
3.2.5.5 dNTP-mix.................................................................................... 46
3.2.5.6 PBS buffer................................................................................... 46
3.2.5.7 Proteinase-K solution.................................................................. 46
3.2.5.8 TBS buffer (10x) (pH7.6)............................................................. 46
3.2.5.9 TE buffer ..................................................................................... 46
3.2.5.10 Buffers for agarose gels.............................................................. 46
3.2.5.10.1 TAE buffer (50x).......................................................................... 46
3.2.5.10.2 TAE running buffer (1x)............................................................... 47
3.2.5.10.3 Loading buffer for DNA (6x) ........................................................ 47
3.2.5.11 Solutions for Southern blot.......................................................... 47
3.2.5.11.1 Denaturation solution .................................................................. 47
3.2.5.11.2 Neutralization solution................................................................. 47
3.2.5.11.3 SSC buffer (20x) (pH 7.0) ........................................................... 47
3.2.5.11.4 Washing solution I....................................................................... 47
3.2.5.11.5 Washing solution II...................................................................... 47
3.2.6 Oligonucleotides ................