-210 Nottingham - Nottingham eTheses - The University of Nottingham

More documents

Recommendations

Info

pFF PKA PKC PMSF PMSG ProMI PVA PVP PVS PZM RFP rpm RNA SA SCNT SDS SDS-PAGE porcine follicular fluid protein kinase A protein kinase C phenylmethylsulphonyl fluoride pregnant mare serum gonadotropin prometaphase of the first meiotic division polyvinylalcohol polyvinylpyrrolidone perivitelline space Porcine Zygote Medium red fluorescent protein rotation per minute ribonucleic acid simultaneous activation somatic cell nuclear transfer sodium dodecyl sulfate sodium dodecyl sulfate polyacrylamide gel electrophoresis second standard error of mean threonine telophase of the first meiotic division telophase of the second meiotic division two-step activation sec SEM T TI TII TA TCM 199 Tissue Culture Medium 199 TEMED N, N, N', N'-tetramethylethylenediamine TL-HEPES HEPES-Buffered TLP Tyrode's Albumin Lactate Pyruvate Medium Tyrode's Lactate Pyruvate Medium TSA trichostatin A 09 microgram µl microlitre µM micromolar sec microsecond UK United Kingdom UV ultraviolet USA United States of America V volt v/v volume : volume W/v weight : volume Y tyrosine xv
CHAPTER 1 LITERATURE REVIEW The aims of this thesis are to establish reliable and reproducible techniques for porcine somatic cell nuclear transfer (SCNT). This literature review will introduce and discuss the cloning technology, in particular, a brief history and introduction to nuclear transfer, biological and technical factors affecting the efficiency of somatic cell cloning (focusing on porcine cloning), nuclear reprogramming and problems with porcine SCNT. 1.1Brief history and introduction to nuclear transfer Cloning means producing asexually genetically identical individuals (clones), which can be strands of DNA, cells in cell culture or organisms (bacteria, plants or animals). Animal cloning includes two fundamentally distinct techniques: embryo splitting and nuclear transfer (Gurdon and Byrne, 2002). Embryo splitting is literally the separation of single blastomeres or the bissection of compact morula or early blastocyst-stage embryos into two or more partial-embryos. Those embryos which survive this manipulation can then develop into genetically identical individuals. Such procedures can be accomplished using micro-needles or razor blades (Prather and First, 1990). The isolated blastomere or split embryos are then transferred into uteri of surrogate mothers for development to term. In contrast, nuclear transfer techniques allow the production of multiple copies of an embryo, foetus or animal. Nuclear transfer is the transfer of a nucleus from a donor cell (karyoplast) into a recipient cell (cytoplast, typically an unfertilised egg) which has had its nuclear genetic material removed. In general, transfer of the nucleus is i
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: MgSO4.7H20 MAPK MAPKK MAPKKK MBP mg
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 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
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,
Page 139 and 140:
and human ovarian oocytes. Nature,
Page 141 and 142:
Funahashi, H., Cantley, T. C. and D
Page 143 and 144:
Hartwell, L. H. (1973) Three additi
Page 145 and 146:
Jelinkovä, L., Kubelka, M., Motlik
Page 147 and 148:
Kubelka, M., Anger, M., Pavlok, A.,
Page 149 and 150:
Le Bourhis, D., Beaujean, N., Ruffi
Page 151 and 152:
Liu, L., Oldenbourg, R., Trimarchi,
Page 153 and 154:
Minshull, J., Blow, J. J. and Hunt,
Page 155 and 156:
Oback, B. and Wells, D. N. Cloning
Page 157 and 158:
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
Page 163 and 164:
Development. 55(2), 121-127. Törne
Page 165 and 166:
Production of the first cloned came
Page 167 and 168:
oocytes. Reproduction, 125(5), 645-
Page 169 and 170:
00 Cl) h 'Cb a) Ü^W wv F NC a b d
Page 171 and 172:
In In ý. ö O V %) ö ö N _ ö O
Page 173 and 174:
- u2 y vý'ý OOU 1-1 00 ^ OO NN ö
Page 175 and 176:
aý yy U a 2 w I-N O N 'T N 1-1 NO
Page 177:
. 10 CA aý v 2 4 1-1 1-1 o Np cc o
show all

-210 Nottingham - Nottingham eTheses - The University of Nottingham

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?