-210 Nottingham - Nottingham eTheses - The University of Nottingham

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1999-2000), ionomycin plus 6-DMAP (Betthauser et al., 2000), A23187 plus CHX (Walters and Wheeler, 2002), etc. Also, physical and chemical activation have been applied in animal cloning. For example, chemical activation has been used in cloning sheep (Loi et al., 2002), cattle (Cibelli et al., 1998) and pigs (Betthauser et al., 2000). Electrical activation has been used for nuclear transfer in cattle (Kato et al., 1998), sheep (Wilmut et al., 1997) and pigs (Polejaeva et al., 2000). To improve the efficiency of SCNT, many related factors that influence development need to be optimised in a species specific manner. However, successful porcine cloning reports have almost all used electrical stimulation which was first reported in mammals in 1970 (Tarkowski et al., 1970). In terms of electrical parthenogenetic activation, only two papers have reported more than 50% of porcine oocytes developing to the blastocyst stage (Zhu et al., 2002; Lee et al., 2004). Zhu et al. (2002) suggested that activation by multiple pulses of lower field strength was beneficial for parthenotes to develop to the blastocyst stage. Lee et al. (2004) reported that a single pulse with calcium rise was sufficient to activate pig oocytes and to achieve a high rate of blastocyst development. However, the best parameters for parthenogenetic blastocyst formation do not always result in the best cloning efficiency (De Sousa et al., 2002; Lee et al., 2003). For successful porcine SCNT, a reliable and reproducible parthenogenetic activation system is required. Also, the method of activation is critical for subsequent development. Although the experiments performed in Chapter 3 demonstrated that cAMP was more effective in synchronising porcine oocyte maturation than CHX, the developmental potential of synchronised oocytes remained to be demonstrated. The production of embryos by SCNT requires the removal of the maternal genetic material, a process termed enucleation. Enucleation has been achieved by both 72
mechanical and chemical procedures. The enucleation methods employed vary between species and cell cycle stage. Cytoplasm of the recipient cells are also removed when the genetic material is removed by enucleation (Dominko et al., 2000). Conventionally, MII enucleation is used for SCNT. At MII stage, the maternal DNA appears as highly condensed chromosomes arranged on a metaphase spindle. The metaphase spindle of MII oocytes is not visible by light microscope because of the presence of cytoplasmic lipid. The blind enucleation is carried out by aspirating the cytoplasm below PBI using PBI as a marker for the location of MII spindle (Chapter 1). Enucleation of oocytes at TI stage has been used successfully in cloning sheep (Chapter 1). In sheep, TI enucleation led to the removal of a significantly smaller karyoplast (15.8 ± 2.4 4m diameter) than enucleation at MII (35.2 ± 3.1 . tm diameter) and consequently removed significantly less of the oocyte cytoplasm (0.2% and 2.3%, respectively; P
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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
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
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: CHAPTER 4 Parthenogenetic developme
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 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,
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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.,
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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
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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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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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