Inducible expression of RANKL in transgenic pigs under the control of the Tet-On system [Elektronische Ressource] / von Eleonore Schilling

Eleonore Schilling

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Aus dem Department für Veterinärwissenschaften
der Tierärztlichen Fakultät der
Ludwig-Maximilians-Universität München

Arbeit angefertigt unter der Leitung von
Univ.-Prof. Dr. E. Wolf


Inducible expression of RANKL in
transgenic pigs under the control of the
Tet-On system




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


von
Eleonore Schilling
aus Herten


München 2011 Gedruckt mit Genehmigung der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München










Dekan: Univ.-Prof. Dr. Braun

Berichterstatter: Univ.-Prof. Dr. Wolf

Korreferent/en: Univ.-Prof. Dr. Dr. habil. Heinritzi











Tag der Promotion:
12. Februar 2011










Meinem Ehemann


Table of contents IV
TABLE OF CONTENTS
TABLE OF CONTENTS ....................................................................................... IV
ABBREVIATIONS ............................. VIII
I. INTRODUCTION .................................................................................. 1
II. LITERATURE ........................ 3
1. Osteoporosis in general ........................................................................... 3
1.1. Pathophysiology of osteoporosis ............................... 3
1.1.1. Definition ................................................................................................. 3
1.1.2. Bone remodeling ...................... 3
1.1.3. Clinical aspects ......................... 3
1.1.4. Classification ............................................................................................ 4
1.1.5. Fragility fractures and health risk .............................. 4
1.2. Economic burden ...................... 5
2. Large animal models for osteoporosis.................................................... 5
2.1. Need for large animal models ................................................................... 5
2.2. Sheep as animal models ............ 6
2.3. Dog as animal models ............................................................................... 7
2.4. Nonhuman primates as animal models ...................... 8
2.5. Pig as animal models ................ 9
2.6. Comparing different large animal models ............................................... 10
3. Relevance of RANKL in health and disease ........ 12
3.1. Role of RANKL/RANK/OPG system in bone remodeling ...................... 12
3.1.1. RANKL/RANK/OPG signaling .............................................................. 12
3.1.2. RANKL .................................................................. 12
3.1.3. RANK .... 13
3.1.4. OPG ....................................... 13
3.2. RANKL/RANK/OPG axis in the state of disease .................................... 15
3.3. sRANKL transgenic mice ....... 16
4. Gene regulation by a tetracycline inducible system ............................ 16
4.1. Inducible gene regulation ........................................................................ 16
4.2. Tet-Off (tTA) .......................................................... 17 Table of contents V
4.3. Tet-On (rtTA) ......................................................................................... 18
4.4. Doxycycline ........................... 19
5. Techniques of transgenesis in pigs ....................... 19
5.1. DNA Microinjection ............................................................................... 19
5.2. Sperm mediated gene transfer . 20
5.3. Lentiviral gene transfer ........... 22
5.4. Somatic cell nuclear transfer (SCNT) ...................................................... 22
6. Examples of genetically modified pigs ................. 24
6.1. Swine in biomedical research .................................................................. 24
6.2. Transgenic human disease models .......................... 24
6.2.1. Human heart disease model .... 24
6.2.2. Cystic fibrosis model .............................................................................. 24
6.2.3. Alzheimer´s disease model ..... 25
6.2.4. Diabetes mellitus type 3 model ............................................................... 25
6.3. Xenotransplantation ................................................................................ 26
7. Embryo transfer (ET) in the pig .......................... 27
7.1. Factors influencing ET success ............................................................... 27
7.2. Pregnancy rates ....................................................... 28
7.3. Endoscopic embryo transfer .................................... 28
III. MATERIALS AND METHODS .......................................................... 29
1. Equipment and expendable items ........................ 29
1.1. In vitro works ......................................................... 29
1.2. Embryo transfer ...................................................... 30
2. Used media and stock solutions ............................ 30
2.1. Stock solutions ....................................................... 30
2.2. Cell culture ............................................................. 31
2.3. Nuclear transfer ...................... 32
3. Establishment of transgenic cell lines .................................................. 34
3.1. Cells ....................................................................... 34
3.2. Transfection of cells ............... 35
3.3. Mass cell selection .................................................................................. 35
4. Vector design......................... 35 Table of contents VI
4.1. Lentiviral vectors .................................................................................... 35
4.2. Conventional vectors .............. 37
5. Procedure of somatic cell nuclear transfer .......................................... 38
5.1. In vitro maturation of oocytes ................................. 38
5.1.1. Ovary collection ..................................................... 38
5.1.2. Oocyte collection .................................................... 38
5.1.3. Selection of oocytes ................ 38
5.1.4. Oocyte maturation .................................................. 39
5.1.5. Denudation of matured oocytes ............................................................... 39
5.2. Nuclear transfer in vivo experiment ........................ 39
5.2.1. Enucleation ............................................................. 39
5.2.2. Donor cell preparation ............................................................................ 40
5.2.3. Donor cell injection ................ 40
5.2.4. Fusion ..................................... 41
5.2.5. Activation ............................................................... 41
5.2.6. In vitro culture of reconstructed embryos ................................................ 41
5.3. Nuclear transfer in vitro experiment ........................ 41
5.4. Embryo transfer ...................................................... 42
5.4.1. Estrus synchronization ............................................ 42
5.4.2. Endoscopic embryo transfer .................................... 42
6. Pregnancy control and birth ................................ 43
6.1. Pregnancy control ................................................................................... 43
6.2. Induction of labor 43
7. Overview: transgenic pig production via SCNT .................................. 43
8. In vivo doxycycline stimulation ............................................................ 44
9. Statistical analysis ................................................. 44
IV. RESULTS.............................................................................................. 46
1. Assessment of SCNT and ET ................................ 46
1.1. Overview of the years 2006 to 2009 ........................................................ 46
1.2. Outcome of in vivo SCNT procedure ...................... 48
1.3. Evaluation in vivo SCNT data................................................................. 49
1.3.1. Impact of seasonal change ...... 49
1.3.2. Effects of different SCNT donor cell treatment ....... 51 Table of contents VII
1.3.3. Different time periods of embryo culture ................................................ 53
1.3.4. Number of transferred NT embryos per recipient .... 55
2. Production of cloned RANKL transgenic pig ...................................... 57
2.1. In vitro SCNT embryo development competence of different cell lines ... 57
2.2. In vivo RANKL and Tet-On SCNT experiments ..... 58
2.2.1. Recovery of Tet-On+RANKL+Neo fetuses ............................................ 60
2.2.2. Recloning of Fetus 3 ............................................... 60
2.2.3. Birth of Tet-On+CAG piglets ................................. 61
2.2.4. Birth of Tet-On 9894