-210 Nottingham - Nottingham eTheses - The University of Nottingham

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ACKNOWLEDGEMENTS I would like to express my sincere respect to my supervisor Professor Keith H. S. Campbell for offering me an opportunity to do my PhD project in his research group, and for his guidance and encouragement during my studies. I would like to thank Dr. Be Zhu, Dr. Inchul Choi, Dr. Joon-Hee Lee, Dr. Kwanta Thansa, Dr. Dasari Amarnath, Dr. Kamila Derecka, Dr. Jinpei Ye, Dr. Joanne Colema, Dr. Emma Spiking, Dr. Ramiro Alberio, Dr. Richard David William Kelly, Dr. Susan Liddell, Mrs. Patricia Fisher, Miss Emma Gregson, Mr. Oliver Brookes, Mr. Adel Moawad, Mr. Leigh Silvester, Mr. Jim Craig for their help and technical guidance during my studies. I would like to thank Dr. Kong Wing, Miss Wioletta Pijacka, Mrs. Shin Chong, Mr. Kunal M Saini, Mr. Ian Smitheram, and all my friends at Sutton Bonington for their advice and friendship. I would like to thank my parents and elder brother for their love, care and support. Also, I would like to thank my previous classmates and friends for their help and encouragement. I would like to express my appreciation to Pro-Vice-Chancellor, Professor Donald Grierson, Fellow of Royal Society to recommend me to Keith H. S. Campbell's lab. I would like to thank the University of Nottingham for U21 scholarship. ill
TABLE OF CONTENTS ABSTRACT ................................................................................. i ACKNOWLEDGEMENTS ......................................................... iii TABLE OF CONTENTS ........................... LIST OF PUBLICATIONS ......................................................... iv ix LIST OF FIGURES ..................................................................... x LIST OF TABLES ..................................................................... xi LIST OF ABBREVIATIONS ...................................................... xii CHAPTER 1 .............................................................................. 1 Literature review 1.1 Brief history and introduction of nuclear transfer 1 1.1.1 .............................. Early experiments of sea urchin and frogs 3 .................................... 1.1.2 Mammalian experiments ......................................................... 5 1.1.3 Why SCNT? ........................................................................ 8 1.1.4 Why are pigs important? 8 1.1.4.1 ......................................................... Fundamental science 8 1.1.4.2 ............................................................... Medical applications 9 1.1.4.3 ............................................................... Agricultural applications 10 ......................................................... 1.2 Biological factors affecting the efficiency of somatic cell cloning......... 11 1.2.1 Recipient cells 11 1.2.1.1 ................................................ ..................... Types of recipient cells 11 1.2.1.2 ............................................................ MPF in oocytes ..................................................................... 11 1.2.1.2.1 MPF and cell cycle .................................................................. 11 1.2.1.2.2 MPF and oocytes 12 1.2.1.3 .................................................................. MAPK in oocytes .................................................................. 13 1.2.2 Donor cells ........................................................................... 16 1.2.3 Coordination of cytoplast-karyoplast cell cycles 17 1.2.3.1 ............................... .. Recipient-donor cell cycle coordination theory 17 1.2.3.2 ................................. MPF, MAPK, NEBD and PCC in recipient-donor coordination 1.2.4 ............. .. 21 Producing cytoplast-karyoplast couplets: genomic reprogramming......... 21 1.2.4.1 Enucleation of the oocytes: maintaining ploidy................................. 21 1.2.4.2 Quiescence of donor cells ................................. 21 1.2.4.3 Fusion ........................ and activation ...................................... iv
Page 1 and 2: The University of -210 Nottingham M
Page 3: treated oocytes was recorded at 38
Page 7 and 8: 2.5.1.2 Preparation of enucleation/
Page 9 and 10: CHAPTER 6 .........................
Page 11 and 12: LIST OF FIGURES Figure 1.1 Process
Page 13 and 14: LIST OF ABBREVIATIONS A23187 AC AI
Page 15 and 16: MgSO4.7H20 MAPK MAPKK MAPKKK MBP mg
Page 17 and 18: CHAPTER 1 LITERATURE REVIEW The aim
Page 19 and 20: The production of multiple offsprin
Page 21 and 22: cytoplasm which contained the nucle
Page 23 and 24: pluripotency, as demonstrated by th
Page 25 and 26: gene-targeting via somatic cell clo
Page 27 and 28: conventional breeding and genetic m
Page 29 and 30: Oocyte maturation is generally cont
Page 31 and 32: ooýcýýommý MPF Time Figure 1.3
Page 33 and 34: not obtain clones by SCNT using bra
Page 35 and 36: A Nuclear NEBD PCC reformation DNA
Page 37 and 38: 1.2.3.2 MPF, MAPK, NEBD and PCC in
Page 39 and 40: Little is known about the mechanism
Page 41 and 42: development of porcine embryos prod
Page 43 and 44: short-wavelength, UV excitable fluo
Page 45 and 46: The procedure is to expel the PBI a
Page 47 and 48: The majority of successful porcine
Page 49 and 50: Until 1997, the established dogma i
Page 51 and 52: deacetylases (HDACs). Trichostatin
Page 53 and 54: Figure 1.5 presents the general exp
Page 55 and 56:
CHAPTER 2 General materials and met
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2.3 Oocyte staining and assessment
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holding pipette enucleation pipette
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! 00-0 !".. ww "'') ^ýýe ;ý ý
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,rjý. 7!: rY Is All Pi D r. Figure
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were washed three times in embryo c
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Three replicates of batches of oocy
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fertilisation, the occurrence of po
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3.2 MATERIALS AND METHODS 3.2.1 Exp
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I 57
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3.3 RESULTS 3.3.1 Conventional matu
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ýIC M 00 N N N N 0 öa oÜd U 0 0
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Meiotic stage in CAMP treated oocyt
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3.4 DISCUSSION The development of N
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44 h. It was comparable to that (93
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oocyte (Chen et al., 1990). In the
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CHAPTER 4 Parthenogenetic developme
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mechanical and chemical procedures.
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COCs were collected and oocytes wer
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f. " ý1403: 0.71 14 - 'Ir I A Figu
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100% 90% 80% 70% d 60% 50% 0 40% 30
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4.4 DISCUSSION For successful porci
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In addition to the listed benefits
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CHAPTER 5 Effect of caffeine treatm
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in vitro substrates. In pigs, Naito
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5.2 MATERIALS AND METHODS 5.2.1 Exp
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5.2.2 In vitro maturation of porcin
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Blastocysts at day 7 were stained a
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Figure 5.2 MPF and MAPK activities
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A 4500000 N 4000000 3500000 Z 30000
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e ýo; +ý \.. 4.0 ob "i "s NW Ir
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5.3.4 Effects of caffeine treatment
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Figure 5.6 Effects of 5 mM caffeine
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5.4 DISCUSSION In the first experim
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In the final experiment, developmen
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CHAPTER 6 General discussion 7.1 Ma
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cumulus cells could be observed spe
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parthenogenetic development excludi
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demonstrated treatment of the TI en
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REFERENCES Abeydeera, L. R., Wang,
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Bromhall, J. D. (1975) Nuclear trna
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Christmann, L., Jung, T. and Moor,
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and human ovarian oocytes. Nature,
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Funahashi, H., Cantley, T. C. and D
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Hartwell, L. H. (1973) Three additi
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Jelinkovä, L., Kubelka, M., Motlik
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Kubelka, M., Anger, M., Pavlok, A.,
Page 149 and 150:
Le Bourhis, D., Beaujean, N., Ruffi
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Liu, L., Oldenbourg, R., Trimarchi,
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Minshull, J., Blow, J. J. and Hunt,
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Oback, B. and Wells, D. N. Cloning
Page 157 and 158:
A. E., Garst, A. S., Moore, M., Dem
Page 159 and 160:
Rossomando, A. J., Payne, D. M., We
Page 161 and 162:
Spemann, H. Embryonic development a
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Development. 55(2), 121-127. Törne
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Production of the first cloned came
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oocytes. Reproduction, 125(5), 645-
Page 169 and 170:
00 Cl) h 'Cb a) Ü^W wv F NC a b d
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In In ý. ö O V %) ö ö N _ ö O
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- u2 y vý'ý OOU 1-1 00 ^ OO NN ö
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aý yy U a 2 w I-N O N 'T N 1-1 NO
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. 10 CA aý v 2 4 1-1 1-1 o Np cc o
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