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1.3.3 Enucleation The purpose of enucleation is to remove the genetic material from the recipient cell. In general, enucleation is achieved by mechanical means (Li et al., 2004), although chemical enucleation has also been reported (Fulka and Moor, 1993). The methods employed vary between species and cell cycle stage. In mouse and rat zygotes, the pronuclei are visible under phase contrast microscopy at MII and the spindle can be observed by use of a Pol-Scope (Liu et al., 2000). In contrast, in cattle, sheep and pig oocytes and zygotes, the cytoplasm is more granular and neither the spindle nor pronuclei are visible. However, pronuclei can be made visible by centrifugation (Tatham et al., 1995). Enucleation of both oocytes and zygotes is generally aided by treating with cytochalasin B or D to destabilise cortical microflaments and therefore facilitate disruption of the plasma membrane (Paterson et al., 2002). Removing the genetic material also removes a portion of the cytoplasm, if the aspirated karyoplast is too large, this may damage the oocyte (Dominko et at, 2000). 1.3.3.1 Metaphase II enucleation Metaphase II (MII) enucleation is carried out by aspirating the ooplasm adjacent to the first polar body (PBI) without DNA staining. It has been successfully used in porcine SCNT. A limitation is that it removes up to 30% of the cytoplasm within an oocyte. In addition, up to 30% of oocytes are not enucleated because the PBI of domestic animal oocytes is frequently displaced from its expected position due to removal of cumulus cells before oocyte manipulation which disrupts the contact between PBI and the MII spindle (Li et al., 2004). 1.3.3.2 Enucleation with Hoechst staining and UV light This approach routinely visualises oocyte chromatin by staining with a 26
short-wavelength, UV excitable fluorochrome such as Hoechst 33342 (Tsunoda et al., 1988) and exposure to UV light method has been applied successfully in porcine cloning. The advantage of visualising the DNA is that very little cytoplasm surrounding the spindle is removed. However, the method poses the risk of damaging the maternal cytoplast by exposure to UV irradiation. However a very short exposure is tolerable as evidenced by the birth of live offspring (Li et al., 2004). 1.3.3.3 Telophase I enucleation The method was first introduced in the mouse (Kono et al., 1991). At telophase of the first meiotic division (TI), extrusion of the first polar body is visible as an extrusion cone on the surface of the oocyte. The meiotic chromosomes have separated but are still attached to the meiotic spindle. A portion of cytoplasm is removed from the extruding region containing the telophase chromosomes and spindle. This method has several advantages over MII enucleation, firstly a smaller volume of oocyte cytoplasm is removed and secondly much higher percentage of enucleated oocytes is achieved (Lee and Campbell, 2006). Ovine clones have been produced by TI enucleation (Choi and Campbell, 2010). This method has potential to be used in porcine SCNT. 1.3.3.4 Telophase II enucleation Telophase II (TII) enucleation is based on the removal of chromatin after oocyte activation. As an extrusion cone is visible allowing aspiration of the telophase second polar body and surrounding cytoplasm are aspirated at the TII stage (Bordignon and Smith, 1998). This method has not been used successfully to obtain porcine clones. A disadvantage of this approach is that the drop in MPF at the telophase stage may affect the cloning efficiency although the enucleation method is efficient (Li et al., 27
Page 1 and 2: The University of -210 Nottingham M
Page 3 and 4: treated oocytes was recorded at 38
Page 5 and 6: 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: development of porcine embryos prod
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 and 88: CHAPTER 4 Parthenogenetic developme
Page 89 and 90: mechanical and chemical procedures.
Page 91 and 92: COCs were collected and oocytes wer
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f. " ý1403: 0.71 14 - 'Ir I A Figu
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100% 90% 80% 70% d 60% 50% 0 40% 30
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4.4 DISCUSSION For successful porci
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In addition to the listed benefits
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CHAPTER 5 Effect of caffeine treatm
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in vitro substrates. In pigs, Naito
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5.2 MATERIALS AND METHODS 5.2.1 Exp
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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
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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