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performed as described in Chapter 2 (section 2.6) For SCNT, TI enucleated oocytes were washed three times in modified NCSU-23 and cultured in modified NCSU-23 until 38 hpm. Then they were washed three times in modified NCSU-23 with 5 mM caffeine and cultured in modified NCSU-23 with 5 mM caffeine until 42 hpm. 5.2.8 Preparation of donor cells Cumulus cells were prepared as described in Chapter 2 (section 2.5.3). 5.2.9 Cell transfer Cell transfer was performed as described in Chapter 2 (section 2.5.4). 5.2.10 Electrofusion As described in Chapter 2 (section 2.5.5), electrofusion was conducted on a heated stage at 39°C immediately after cell transfer. 5.2.11 Activation Activation was performed as described in Chapter 2 (section 2.7). For parthenogenetic activation, oocytes were denuded of cumulus cells at 36 hpm by vortexing with hyaluronidase for 5 min and treated with caffeine until 44 hpm. Oocytes were selected and activated. For SCNT, cytoplast-karyoplast couplets were selected and activated. 5.2.12 In vitro culture of porcine embryos Following activation, oocytes or couplets were washed and then cultured as described in Chapter 2 (section 2.8). 5.2.13 Embryo staining and evaluation of development 92
Blastocysts at day 7 were stained and evaluated as described in Chapter 2 (section 2.9). 5.2.14 Statistical analysis At least three replicates of batches of oocytes were carried out for MPF/MAPK double kinase assay and parthenogenetic activation. Data was analysed by chi-square test, T-test or ANOVA. ANOVA was performed using Genestat version 12. Probabilities ofp < 0.05 were considered statistically different. 93
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The University of -210 Nottingham M
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treated oocytes was recorded at 38
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TABLE OF CONTENTS ABSTRACT ........
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2.5.1.2 Preparation of enucleation/
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CHAPTER 6 .........................
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LIST OF FIGURES Figure 1.1 Process
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LIST OF ABBREVIATIONS A23187 AC AI
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MgSO4.7H20 MAPK MAPKK MAPKKK MBP mg
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CHAPTER 1 LITERATURE REVIEW The aim
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The production of multiple offsprin
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cytoplasm which contained the nucle
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pluripotency, as demonstrated by th
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gene-targeting via somatic cell clo
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conventional breeding and genetic m
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Oocyte maturation is generally cont
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ooýcýýommý MPF Time Figure 1.3
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not obtain clones by SCNT using bra
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A Nuclear NEBD PCC reformation DNA
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1.2.3.2 MPF, MAPK, NEBD and PCC in
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Little is known about the mechanism
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development of porcine embryos prod
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short-wavelength, UV excitable fluo
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The procedure is to expel the PBI a
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The majority of successful porcine
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Until 1997, the established dogma i
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deacetylases (HDACs). Trichostatin
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Figure 1.5 presents the general exp
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CHAPTER 2 General materials and met
Page 57 and 58: 2.3 Oocyte staining and assessment
Page 59 and 60: holding pipette enucleation pipette
Page 61 and 62: ! 00-0 !".. ww "'') ^ýýe ;ý ý
Page 63 and 64: ,rjý. 7!: rY Is All Pi D r. Figure
Page 65 and 66: were washed three times in embryo c
Page 67 and 68: Three replicates of batches of oocy
Page 69 and 70: fertilisation, the occurrence of po
Page 71 and 72: 3.2 MATERIALS AND METHODS 3.2.1 Exp
Page 73 and 74: I 57
Page 75 and 76: 3.3 RESULTS 3.3.1 Conventional matu
Page 77 and 78: ýIC M 00 N N N N 0 öa oÜd U 0 0
Page 79 and 80: Meiotic stage in CAMP treated oocyt
Page 81 and 82: 3.4 DISCUSSION The development of N
Page 83 and 84: 44 h. It was comparable to that (93
Page 85 and 86: oocyte (Chen et al., 1990). In the
Page 87 and 88: CHAPTER 4 Parthenogenetic developme
Page 89 and 90: mechanical and chemical procedures.
Page 91 and 92: COCs were collected and oocytes wer
Page 93 and 94: f. " ý1403: 0.71 14 - 'Ir I A Figu
Page 95 and 96: 100% 90% 80% 70% d 60% 50% 0 40% 30
Page 97 and 98: 4.4 DISCUSSION For successful porci
Page 99 and 100: In addition to the listed benefits
Page 101 and 102: CHAPTER 5 Effect of caffeine treatm
Page 103 and 104: in vitro substrates. In pigs, Naito
Page 105 and 106: 5.2 MATERIALS AND METHODS 5.2.1 Exp
Page 107: 5.2.2 In vitro maturation of porcin
Page 111 and 112: Figure 5.2 MPF and MAPK activities
Page 113 and 114: A 4500000 N 4000000 3500000 Z 30000
Page 115 and 116: e ýo; +ý \.. 4.0 ob "i "s NW Ir
Page 117 and 118: 5.3.4 Effects of caffeine treatment
Page 119 and 120: Figure 5.6 Effects of 5 mM caffeine
Page 121 and 122: 5.4 DISCUSSION In the first experim
Page 123 and 124: In the final experiment, developmen
Page 125 and 126: CHAPTER 6 General discussion 7.1 Ma
Page 127 and 128: cumulus cells could be observed spe
Page 129 and 130: parthenogenetic development excludi
Page 131 and 132: demonstrated treatment of the TI en
Page 133 and 134: REFERENCES Abeydeera, L. R., Wang,
Page 135 and 136: Bromhall, J. D. (1975) Nuclear trna
Page 137 and 138: Christmann, L., Jung, T. and Moor,
Page 139 and 140: and human ovarian oocytes. Nature,
Page 141 and 142: Funahashi, H., Cantley, T. C. and D
Page 143 and 144: Hartwell, L. H. (1973) Three additi
Page 145 and 146: Jelinkovä, L., Kubelka, M., Motlik
Page 147 and 148: Kubelka, M., Anger, M., Pavlok, A.,
Page 149 and 150: Le Bourhis, D., Beaujean, N., Ruffi
Page 151 and 152: Liu, L., Oldenbourg, R., Trimarchi,
Page 153 and 154: Minshull, J., Blow, J. J. and Hunt,
Page 155 and 156: Oback, B. and Wells, D. N. Cloning
Page 157 and 158: A. E., Garst, A. S., Moore, M., Dem
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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-
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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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