Aus dem Department für Veterinärwissenschaften der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Arbeit angefertigt unter der Leitung von Univ.-Prof. Dr. Eckhard Wolf (Re)producing transgenic pigs for xenotransplantation – selection of founder animals and establishment of breeding herds Inaugural Dissertation zur Erlangung der tiermedizinischen Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München von Andrea Bähr aus München München 2011 Gedruckt mit der Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. Braun Berichterstatter: Univ.-Prof. Dr. Wolf Korreferent: Univ.-Prof. Dr. Zerbe Tag der Promotion 30. Juli 2011 Für den Puck TABLE OF CONTENTS 1 INTRODUCTION ......................................................................................1 2 REVIEW OF THE LITERATURE ..........................................................3 2.1 The pig as a model species in biomedical research ................................ 3 2.2 Established pig models .............................................................................. 4 2.2.1 Neurodegenerative diseases ........................................................................ 4 2.2.2 Cardiovascular diseases ............................................................................... 6 2.2.3 Diabetes mellitus ......................................................................................... 7 2.2.4 Cystic Fibrosis ............................................................................................. 9 2.3 Pigs as donor animals in the context of xenotransplantation .............. 11 2.3.1 Hyperacute xenograft rejection ................................................................. 12 2.3.2 Acute humoral xenograft rejection ............................................................ 14 2.3.3 Cell-mediated xenograft rejection ............................................................. 16 2.4 (Re)producing transgenic pigs ............................................................... 17 2.4.1 Pig breeds and pig breeding ...................................................................... 19 2.4.2 Inbreeding depression ............................................................................... 22 2.4.3 Inbreeding in pig livestock and experimental animals .............................. 24 2.5 Selection of breeding material – expression analysis ........................... 28 3 ANIMALS, MATERIALS AND METHODS ........................................33 3.1 Animals ..................................................................................................... 33 3.1.1 GalKO ....................................................................................................... 33 3.1.2 CD46 ......................................................................................................... 33 3.1.3 hTM ........................................................................................................... 34 3.1.4 HLA-E ....................................................................................................... 34 3.1.5 LEA29Y .................................................................................................... 35 3.2 Materials .................................................................................................. 36 3.2.1 Chemicals .................................................................................................. 36 3.2.2 Consumables ............................................................................................. 37 3.2.3 Devices ...................................................................................................... 38 3.2.4 Antibodies, drugs, enzymes, oligonucleotides, standards ......................... 39 I Table of contents 3.2.4.1 Antibodies ................................................................................................. 39 3.2.4.2 Drugs ......................................................................................................... 39 3.2.4.3 Enzymes .................................................................................................... 39 3.2.4.4 Oligonucleotides ........................................................................................ 40 3.2.4.5 Protein standards ....................................................................................... 42 3.2.5 Buffers, media, solutions ........................................................................... 42 3.2.6 Kits ............................................................................................................ 48 3.2.7 Others ........................................................................................................ 48 3.2.8 Software .................................................................................................... 48 3.3 Methods .................................................................................................... 49 3.3.1 Genomic analysis ...................................................................................... 49 3.3.1.1 Genotyping of founder animals and F1 generation ................................... 49 3.3.1.2 Duplex PCR ............................................................................................... 51 3.3.1.3 Agarose Gel Electrophoresis ..................................................................... 52 3.3.1.4 Southern Blot ............................................................................................. 52 3.3.1.5 Inverse Polymerase Chain Reaction (inverse PCR) .................................. 56 3.3.1.5.1 Genomic DNA isolation ............................................................................ 57 3.3.1.5.2 DNA fragmentation ................................................................................... 57 3.3.1.5.3 Circularisation of DNA fragments ............................................................ 58 3.3.1.5.4 Nested PCR ............................................................................................... 59 3.3.1.5.5 DNA Eluation ............................................................................................ 62 3.3.1.5.6 Ligation ..................................................................................................... 62 3.3.1.5.7 Heat Shock Transformation ...................................................................... 63 3.3.1.5.8 Plasmid preparation ................................................................................... 63 3.3.1.5.9 Restriction digest ....................................................................................... 64 3.3.1.5.10 PEG precipitation ...................................................................................... 64 3.3.1.5.11 Sequencing ................................................................................................ 65 3.3.1.5.12 EtOH precipitation .................................................................................... 66 3.3.2 Expression analysis ................................................................................... 66 3.3.2.1 Protein isolation ......................................................................................... 67 3.3.2.2 Enzyme-Linked Immunosorbent Assay (ELISA) ..................................... 67 II Table of contents 3.3.2.3 Immunohistochemistry .............................................................................. 69 3.3.3 Calculation of inbreeding coefficient ........................................................ 70 3.3.4 Design of breeding schedules .................................................................... 71 4 RESULTS ..................................................................................................73 4.1 Identification of suitable breeding herds .............................................. 73 4.2 Selection of founder animals .................................................................. 74 4.3 Expression and functional analysis of novel transgenic lines .............. 75 4.3.1 hTM ........................................................................................................... 75 4.3.1.1 Clarification of hTM transgene integration site ........................................ 79 4.3.2 INS-LEA ................................................................................................... 83 4.3.3 CAG-LEA ................................................................................................. 88 4.4 Breeding schedules .................................................................................. 92 4.5 Breeding & analysis of intermediate (F1) generations ........................ 99 4.6 F2 generations and hTM incorporation into breeding herd ............. 108 4.7 Identification of transgene zygosity ..................................................... 109 4.7.1 GalKO duplex PCR ................................................................................. 109 4.7.2 hTM duplex PCR .................................................................................... 111 5 DISCUSSION .........................................................................................113 6 SUMMARY .............................................................................................125 7 ZUSAMMENFASSUNG .......................................................................129 8 INDEX OF FIGURES ............................................................................133