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the microforge and a glass spike added. The pipette was positioned horizontally and bent about 2 cm from the tip at an angle of 25°, thus allowing the tip to be parallel to the manipulation chamber when mounted on the micromanipulator connected to the microscope (Figure 2.1). 2.5.1.3 Manipulation chamber A 90 mm petri dish was used as a manipulation chamber (Figure 2.1). Two parallel columns of three drops (50 µl each) of enucleation medium containing BSA-free NCSU 23 medium supplemented with 1 gM glutathione, 1 mM cysteine, 5 mg/I insulin, 10 ng/ml EGF, 1% essential and 0.5% nonessential amino acids, 10% FBS (Hyclone), 0.05 M sucrose (Fisher Scientific) and 7.5 µg/ml cytochalasin B (CB) and cell transfer medium containing BSA-free NCSU 23 medium supplemented with 1 gM glutathione, 1 mM cysteine, 5 mg/I insulin, 10 ng/ml EGF, 1% essential and 0.5% nonessential amino acids and 10% FBS (Hyclone) were made with two 130 µl drops of PBS containing 10% PVP in the petri dish. The drops were covered in warm (37 °C) mineral oil. The chamber was placed onto the microscope fitted with micromanipulator for oocyte manipulation. 2.5.2 TI oocyte enucleation A portion of cytoplasm containing the extruding TI spindle was aspirated from the oocytes at 36-38 hpm using a 20-25 µm glass micropipette in enucleation medium (Figure 2.2). For enucleation, conventional and/or laser assisted methods were used. Laser assisted enucleation was similar to traditional enucleation except the zona pellucida was cut using a laser (XYclone laser, Hamiltom Thorne, USA) instead of a sharp pipette. The extruding polar body was positioned at 3-o'clock, once aligned, the oocyte was firmly held by suction on holding pipette and the zona pellucida was penetrated by nuclear transfer pipette. To remove the oocyte DNA, a small amount of cytoplasm from the extrusion zone containing the spindle, was aspirated. Enucleation was confirmed by visualisation of DNA in the karyoplast by 5 min Hoechst 33342 44
! 00-0 !".. ww "'') ^ýýe ;ý ý ý. a ` 1.. ' -'i .. v A -B ý... ,.,, "144 C D Figure 2.2 Enucleation of porcine oocytes at telophase of first meiotic division (TI) A) A TI arrested oocyte was held prior to enucleation; B) The same TI arrested oocyte was held prior to enucleation (black arrow: the extruding TI spindle); C) Zona pellucida was broken by enucleation/ nuclear transfer pipette with a spike; D) Enucleated oocytes. Scale bar = 100 pm. 45
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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/
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 and 58: 2.3 Oocyte staining and assessment
Page 59: holding pipette enucleation pipette
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
Page 109 and 110: 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
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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In In ý. ö O V %) ö ö N _ ö O
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aý yy U a 2 w I-N O N 'T N 1-1 NO
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