The Influence of Oxidative Stress, Carcinogens and Cloning on DNA ...

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Introduction 1.2.7.1 <strong>Cloning</strong> efficiency <strong>The</strong> birth <strong>of</strong> the first mammal cloned from an adult donor cell, ‘Dolly’, proved the success <strong>of</strong> cloning (125) <strong>and</strong> has initiated a stream <strong>of</strong> researches to improve cloning technology. However the overall efficiency <strong>of</strong> this technology is low (126-128) noting a success rate ranging from 0.1 % to 3 % with an exception <strong>of</strong> 15-20 % success recorded by cattle as a model extensively used for mammalian cloning (129) . It was estimated that the clones show frequently high mortality rates in utero due to the incomplete reprogramming <strong>of</strong> nuclear activities (127) . Normally, somatic nuclei acquire highly specialized DNA <strong>and</strong> chromatin modifications during the differentiation stage in order to support early development (130) . Upon somatic nuclear transfer into oocytes, the donor nucleus should be remodelled from highly differentiated somatic patterns to a totipotent embryonic pattern (zygote that can develop into any sort <strong>of</strong> cell) then to pluripotent pattern (embryo state that gains the capacity to develop to many cell types). <strong>The</strong> end <strong>of</strong> these development stages is referred to ‘terminal differentiation’ when cells gain specific characteristics that allow them to do a specific function. This process is called ‘epigenetic reprogramming’ occurring in mammalian embryos at two times during their development, once soon after fertilization known as preimplantation reprogramming <strong>and</strong> the other takes place during gametogenesis <strong>and</strong> known as gametogenic reprogramming (Fig. 6). It is usually associated with some changes in epigenetic features similar to those that occur Animal donating oocyte Nucleus removal Animal to be cloned Surrogate mother Enucleated egg Fusion Egg fused with cell Morula Preimplantation reprogramming reprogramming Zygote 2-cell stage 4-cell stage Figure 6: Overview <strong>of</strong> epigenetic reprogramming in cloning process <strong>and</strong> embryo development (Figure modified from www.fda.gov/ucm/groups) 12 Fertilized egg 8-cell stage Terminal differentiation Morula Embryo blastocyst Primary gametes Gametogenic Gametogenic reprogramming
13 Introduction in normal embryos during early development (131, 132) . However, different studies showed an evidence that incomplete or inappropriate epigenetic reprogramming <strong>of</strong> donor cell nuclei is the main cause behind the low cloning efficiency, high embryonic <strong>and</strong> foetal losses after SCNT <strong>and</strong> developmental abnormalities (133, 134) . At different stages, inappropriate reprogramming occurs leading to failure <strong>of</strong> cloning. Simply talking, other logical reasons rather than the failure <strong>of</strong> epigenetic reprogramming can explain the unsuccessful cloning procedure in the embryonic stage. <strong>The</strong>se reasons could be the incompatibility between the enucleated egg <strong>and</strong> the transferred nucleus, or an egg with a newly transferred nucleus may fail to divide or develop properly, or simply the implantation <strong>of</strong> the embryo into the surrogate mother might be impossible. On the other side, the surviving clones can exhibit congenital defects (135) such as LOS (126) , kidney or brain malformations <strong>and</strong> impaired immune systems. 1.2.7.2 Effect <strong>of</strong> cloning on epigenetics High rates <strong>of</strong> embryonic <strong>and</strong> foetal mortality (127, 136) , <strong>and</strong> an increased incidence <strong>of</strong> congenital defects (135, 136) have been associated with perturbations in developmentally important epigenetic marks (127) such as DNA methylation <strong>and</strong> histone modifications (137-139) . <strong>The</strong> methylation <strong>of</strong> DNA is involved in long-term epigenetic silencing <strong>of</strong> specific sequences (43) , including transposons * , imprinted genes <strong>and</strong> pluripotency-associated genes in the cloning process (140) . Various studies had demonstrated that SCNT procedure can impact on the global DNA methylation status <strong>of</strong> cloned bovine embryos by insufficient active de-methylation <strong>of</strong> the nuclear donor DNA after transplantation, by delayed or attenuated further passive de-methylation, <strong>and</strong> by precocious de novo methylation before the 16-cell stage (134, 137, 141, 142) . Other studies had revealed aberrations in global DNA methylation status <strong>of</strong> cloned embryos <strong>and</strong> fetuses in different species (137, 143-145) . In general, an increase in DNA methylation level in cattle clones has been correlated with poor developmental potential <strong>of</strong> cloned preimplantation embryos (137) <strong>and</strong> with compromised foetal overgrowth (144) . <strong>The</strong> global changes in methylation states have also been described for specific genes <strong>and</strong> non-coding DNA sequences under epigenetic control (127, 133, 138, 139, 142, 146) . Although the overview <strong>of</strong> these reports shows a relation between the aberrant methylation level <strong>and</strong> the poor success for the cloning process, the genomic methylation status <strong>of</strong> live <strong>of</strong>fspring was not studied so far.
Page 1 and 2: The Influe
Page 3 and 4: The work at h<stro
Page 5 and 6: First, I feel always grateful to Al
Page 7: I would like to extend my thanks to
Page 11 and 12: The following acro
Page 13 and 14: FDA Food and drug
Page 15: RIA Radioimmunoassay ROS Reactive o
Page 18 and 19: Index 3.1 Spectrophotometeric deter
Page 20 and 21: Index 5.2.4.2 Dialysis of</
Page 22 and 23: Introduction 1.1.2 DNA methylation
Page 24 and 25: Introduction 1.2 The</stron
Page 26 and 27: Introduction Figure 4: Model for me
Page 28 and 29: Introduction Cancer is classified i
Page 30 and 31: Introduction DNA methylation Gene h
Page 34 and 35: Introduction 1.2.8 Twins an
Page 36 and 37: Introduction chemicals, alkylating
Page 38 and 39: Introduction AP sites are not only
Page 40 and 41: Introduction 1.3.2.1 Oxidat
Page 42 and 43: Introduction Finally, flavo- <stron
Page 44 and 45: Introduction chromatography with el
Page 46 and 47: Introduction One of</strong
Page 48 and 49: Introduction facturing of</
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Page 52 and 53: Introduction HO O O Figure 15: Mech
Page 54 and 55: Introduction - Methylation Sensitiv
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Page 58 and 59: Introduction - Radioactive-mediated
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Results and Discus
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Summary 4 Summary DNA methylation i
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Summary The same s
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Experimental part Sodium tetrapheny
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Experimental part � Sodium tetrab
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Experimental part framework was fas
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Experimental part propanol
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Experimental part 12.5 mU/µL SPD)
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Experimental part Condition Value D
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Experimental part 5.4 Instrumentati
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Experimental part Graduated cylinde
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Experimental part 3-Nitrobenzanthro
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Appendix In-vitro: Denoting a react
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Appendix Laboratory Document Hydrol
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Appendix BODIPY and</strong
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Appendix Description of</st
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Appendix Transmission [%] 120 100 8
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Appendix Figure 62: Mass spectrum E
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List of figures Fi
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List of tables 8 L
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Literature Literature 1. Egger, G.;
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Literature 41. Bestor, T.H.; "<stro
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Literature 78. Venitt, S.; "Mechani
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Literature 113. Sinclair, K.D.; Mce
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Literature 148. Kadan-Lottick, N.S.
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Literature cyte DNA by gas chromato
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Literature 221. Mirand</str
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Literature 257. Lingaraju, G.M.; Sa
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Literature different tissues <stron
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Literature 324. Vishwanath, B.S.; F
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Literature 359. Wei, L.; Li, R.K.;
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Literature 394. Ramsahoye, B.H.; "M
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Literature 431. Wirtz, M.; Schumann
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Literature 467. Cornelius, M.; Wort
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Literature 502. Issa, J.P.J.; Ottav
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Literature 536. Pinheiro, J. <stron
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