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nuclear transfer. Also, effects of caffeine treatment on MPF and MAPK activities in TI enucleated porcine oocytes were evaluated. In addition, the development of SCNT embryos reconstructed using TI enucleated oocytes treated with the optimal concentration of caffeine was recorded. Mammalian cells can be synchronised at various cell cycle stages physically and chemically. Induction of quiescence by serum starvation (BÜrk, 1966) or contact inhibition (Nilausen and Green, 1965) are the most popular methods for donor cell synchronisation in SCNT. Cumulus cells have been shown to be mainly arrested at GO/G I stage without treatment (Schuetz et al., 1996). In these studies, cumulus cells were selected as donor cells for SCNT. 88
5.2 MATERIALS AND METHODS 5.2.1 Experimental design In the first experiment, an MPF/MAPK assay system was to be further developed by modification of methods by Ye et al. (2003). Effects of different media for washing oocytes prior to storing samples at - 80°C on MPF/MAPK activities in porcine oocytes were determined. Effects of different media or solution for washing oocytes prior to storing samples at - 80°C on MPF/MAPK activities in porcine oocytes were determined. These media or solution included modified NCSU-23 medium, collection buffer containing 6.4 mM EDTA (PH7.4), 10 mM NaF and 100 mM Na3VO4 in DPBS and DPBS containing 0.1% PVA. The next two experiments were to select a suitable caffeine concentration according to the effects of different concentration on MPF and MAPK activities and survival development of parthenogenetic embryos. In the second experiment, COCs were stripped of cumulus cells at 36 hpm. Denuded TI and early MII oocytes were selected and cultured in maturation medium (modified NCSU-23) until 38 hpm. Groups of approximately 20 oocytes arrested at TI or early MII stage were cultured in 50 Al modified NCSU-23 with different concentrations of caffeine (0,5,10 and 20 mM) for 6h until 44 hpm and then MPF and MAPK activities at 44 hpm were measured. As control, kinase activities were assayed in denuded intact MII oocytes without caffeine treatment at 44 hpm. Three replicates of batches of oocytes were performed. In the third experiment, COCs were stripped of cumulus cells at 36 hpm and selected TI and early MII oocytes were cultured in HEPES-NCSU-23 until 38 hpm. Groups of approximately 20 oocytes arrested at TI or early MII stage were cultured in 50 . tl maturation medium (modified NCSU-23) with different concentrations of caffeine (0, 5,10 and 20 mM) for 6h until 44 hpm and then activated. Cleavage, blastocyst development and total cell numbers of blastocyst stage embryos were evaluated. Four replicates of batches of oocytes were performed. 89
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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
Page 53 and 54: Figure 1.5 presents the general exp
Page 55 and 56: 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: in vitro substrates. In pigs, Naito
Page 107 and 108: 5.2.2 In vitro maturation of porcin
Page 109 and 110: Blastocysts at day 7 were stained a
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,
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Oback, B. and Wells, D. N. Cloning
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A. E., Garst, A. S., Moore, M., Dem
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Rossomando, A. J., Payne, D. M., We
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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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