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(Wakayama et al., 1998; Onishi et al., 2000). This protocol is quicker as compared to eletrofusion method and it also minimises the amount of somatic cell cytoplasm introduced into enucleated oocytes, which might influence development. 1.3.4.2 Serial transfer The first cloned pigs in the world were produced by serial transfer. The donor cell is inserted into the perivitelline space of an in vivo derived, MII enucleated oocyte, then fused and activated by electrical pulses. Successful fused embryos are cultured overnight. The second nuclear transfer is performed by removing the "pseudo" pronucleus from the reconstructed embryo and fusing the karyoplast to an in vivo fertilised zygote (previously enucleated by removal of the pronuclei). This method helped bypass the inefficiencies of artificial activation procedures and promote more development (Polejaeva et al., 2000). 1.3.4.3 Zona free nuclear transfer This method was developed to avoid the requirement for expensive micromanipulation equipments. Recipient oocytes are halved with a microblade and two enucleated halves are fused together with the donor cell. Piglets have been cloned by this method (Lagutina et al., 2006). 1.3.5 Parthenogenetic activation Fused or reconstructed embryos should be activated to initiate development. Technically, activation protocols include: (1) physical electroporation, (2) calcium ionophores, (3) stronitium, (4) ethanol, (5) protein kinase inhibitors, e. g. ionomycin plus 6-dimethylaminopurine (6-DMAP) or ionomycin plus butyrolactone I, (6) protein synthesis inhibitors, e. g. CHX, electroporation plus CHX, ethanol plus CHX, A23187 plus CHX or ionomycin plus CHX and (7) preactivation of oocytes (Machaty, 2006). 30
The majority of successful porcine cloning reports to date induced activation by electroporation. It can be divided into delayed activation (DA) method, simultaneous activation (SA) method and two-step activation (TA) method. DA method induces electric stimulation (with calcium) 1-1.5 h after fusion (without calcium). SA method induces fusion and activation simultaneously with calcium. TA method induces egg activation in two steps, both with calcium. 1.3.6 Culture and transfer of reconstructed embryos Reconstructed embryos can be cultured to a stage suitable for transfer to a surrogate recipient or transferred directly after reconstruction to the surrogate. The choice of methods is dependent upon species and culture methods. Cattle and sheep are not litter-bearing and it is normal to transfer a small number of blastocyst stage embryos. In contrast, pigs are litter bearing and large numbers of reconstructed zygotes can be transferred. The advantage of direct transfer is that culture conditions in the oviduct are optimal. In cattle and sheep, embryo culture prior to transfer has been carried out in the ligated oviduct of a ewe for 7 days (Willadsen et al., 1986) or in vitro (Cibelli et al., 1997). In pigs, it is not necessary to use temporary recipients because large numbers of embryos need to be transferred. It is also costly to use a temporary recipient as compared to the in vitro culture method. The development of embryos is influenced by cytoplasmic maturation of oocytes and the type of culture system. The factors affecting the culture system include macromolecules such as albumin, carbon dioxide concentration, oxygen concentration, medium renewal, temperature, pH, type of medium overlay, incubation volume, embryo density per volume, culture vessel, air quality, technical diligence and quality control (Gardner and Lane, 2004). A large number of porcine embryo culture media have been used including: modified Whitten's medium (mWM; Beckmann and Day, 1993), North Carolina State 31
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 and 42: development of porcine embryos prod
Page 43 and 44: short-wavelength, UV excitable fluo
Page 45: The procedure is to expel the PBI a
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
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
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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
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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
Page 159 and 160:
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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