-210 Nottingham - Nottingham eTheses - The University of Nottingham

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transferred into 500 µl modified NCSU-23 containing 0.05 M sucrose (Fisher Scientific) for selection at 39°C. Oocytes with an extrusion cone or polar body were regarded as TI or early MIT oocytes. Selection of TI oocytes was also carried out in enucleation medium in the microinjection chamber directly after cumulus removal. 2.5 Nuclear transfer (Campbell et al., 2006; Polejaeva et al., 2005) 2.5.1 Instruments for micromanipulation 2.5.1.1 Preparation of holding pipettes A 1.0 mm (o. d. ) x 0.58 mm (i. d. ) x 10 cm glass capillary (Intracel, England) was pulled by hand over a small flame to make a long parallel length of glass of approximately 150 mm. The pulled capillary was cut using a diamond pencil to give a pipette with a 100-150 µm outer diameter (Figure 2.1). The end of the pipette was fire polished over the filament of the microforge to close the end to a diameter of approximately 25 µm. Finally, the pipette was positioned horizontally and bent about 1 cm from the tip at an angle of 30°, thus allowing the tip to be parallel to the manipulation chamber when mounted in the micromanipulator (Burleigh Instruments Inc., UK) attached to the microscope (Leica DMIRBE, Heidelberg, Germany). 2.5.1.2 Preparation of enucleation/nuclear transfer pipettes A 1.0 mm (o. d. ) x 0.80 mm (i. d. ) x 10 cm glass capillary (Intracel, England) was pulled using a moving-coil microelectrode puller (P-97, Sutter Instruments Co., USA) to give a inner diameter of slightly more than the required diameter (e. g. 20 gm). The capillary was mounted in the microforge and broken at the required diameter by fusing the glass onto the glass bead on the microforge (MF-830, Narishige, Japan) and turning off the heat while drawing it away. The pipette was then ground using a microgrinder (EG-400, Narishige, Japan) at an angle of 45°. The tip was mounted in 42
holding pipette enucleation pipette 300 25° 1cni ---ý, 2cm 25 jun 20 lull t fi PBS/10% PVP cell transfer medium ent)cleatiou meditmi Figure 2.1 Diagram of micromanipulation Instruments and manipulation set up The holding pipette was bent about 1 cm from the tip at an angle of 30° and its end has an inner diameter of approximately 25 Nm. The enucleation pipette was bent about 2 cm from the tip at an angle of 25° and its end has an inner diameter of approximately 20 pm. A manipulation chamber was made from two parallel columns of three drops (50 p1 each) of enucleation medium and cell transfer medium and 130 pi drops of PBS containing 10% PVP in the petri dish. 43
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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 ........
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
Page 57: 2.3 Oocyte staining and assessment
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 and 108: 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.,
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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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In In ý. ö O V %) ö ö N _ ö O
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