List of abbreviations and measurement units

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Chapter 1. General Introduction

1

Chapter 2. Literature Review

3

2.1 The mammalian mitochondrial genome 3

2.1.1 Gene expression 4

2.1.1.1 Gene transcription 5

2.1.1.2 RNA processing 8

2.1.1.3 Translation 9

2.1.2 Replication and maintenance of mtDNA 12

2.2 Human mitochondrial diseases 17

2.2.1 Mitochondrial defects related to mtDNA mutation/rearrangements 17 2.2.2 Disease manifestations: threshold level and complementation 18

2.3 Animal models of mitochondrial disease 20

2.3.1 Mitochondrial transgenic mice 20

2.3.2 Methods for the generation of transmitochondrial transgenic mice 23 2.3.3 Model systems for transmitochondrial mice 25

2.4 Gene targeting of mitochondria 27

2.4.1 Mitochondrial DNA constructs 27

2.4.2 Mitochondrial internalization of nucleic acids 29

2.4.2.1 Mitochondrial compartments 30

2.4.2.2 Import of rRNA and tRNA 31

2.4.2.3 How can nucleic acids be targeted to mitochondria? 32

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2.4.2.3.2 Cytoplasmic factors 34

2.4.2.3.3 Conformation changes 35

2.4.2.3.4 Mitochondrial protein complexes facilitate import? 35

2.5 Mitochondrial DNA delivery systems 37

2.5.1 Targeting molecules into mitochondria 38 2.5.2 Liposome mediated import of exogenous DNA 40

2.5.3 DQAsome 42

2.5.3.1 Micellar form of dequalinium 42

2.5.3.2 Cytotoxicity of dequalinium 43

2.5.3.3 Dequalinium induces a selective loss of mtDNA. 43

2.5.3.4 Dequalinium unwinds DNA 44

2.5.3.5 Effect of dequalinium on cytochrome c oxidase activity 45

2.5.3.6 Effect of culture medium on dequalinium cytotoxicity 45

2.6 Scope of thesis 46

Chapter 3. General Materials and Methods

47

3.1 DNA 47

3.1.1 DNA purification 47

3.1.1.1 Extractionof DNA fragments from agarose gels 47

3.1.1.2 Purification of PCR products 48

3.1.1.3 Minipreps 48

3.1.1.4 Maxipreps 49

3.1.2 Ligation 51

3.1.3 E. coli transformation and plasmid propagation 51

3.1.4 Glycerol stock 52

3.1.5 DNA sequencing 52

3.2 Cell culture 53

3.2.1 Cell lines 53

3.2.2 Culture conditions 53

3.2.3 Cell harvesting 54

3.2.4 Freezing and thawing of cell stocks 55

3.2.5 Assays for cell viability 55

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3.3.1 Transfection reagent 56

3.3.1.1 DQAsome preparation 56

3.3.1.2 Plasmid vectors 57

3.3.1.3 Preparation of lipoplex (liposome-DNA) 57

3.3.2 Transfection assays 58

3.3.2.1 Lipofectin protocols for transient transfection 58

3.3.2.1 DQAsome protocols for transfection 58

3.4 Mitochondria isolation 59

3.5 PCR protocols 60

3.5.1 General PCR 60

3.5.2 PCR for cellular pmtGFP DNA detection. 61

3.5.3 RT-PCR for mtgfp transcripts 62

3.6 Site directed mutagenesis 63

3.6.1 Basic principle of site directed mutagenesis 63

3.6.2 Mutagenesis primer design 64

3.6.3 Mutant strand synthesis 64

3.6.4 Ligation of PCR product 65

3.6.5 Dpn I digestion of the plasmid template 65 3.6.6 Transformation of ultracompetent cells 65

3.7 Flow cytometry 66

3.8 Microscopy 66

3.8.1 Epifluorescence microscopy 66

3.8.2 Confocal microscopy 66

3.9 Freeze-thaw-cultured of transfected cells 67

3.10 Fluorescence in Situ Hybridization (FISH) 67

3.10.1 Preparation of the hybridization probe 67

3.10.2 Nick translation 68

3.10.3 Purification of amine-modified DNA 68

3.10.4 Labeling with a reactive fluorescent dye 69

3.10.5 In situ hybridization 70

3.10.6 Hoechst 33342 staining 71

3.11 Animals 71

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3.11.2 Strains 71

3.11.3 Husbandry (female and male) 71

3.11.4 Embryo 72

3.11.4.1 Superovulation 72

3.11.4.2 Mating 72

3.11.4.3 Embryo collection and cumulus cells removal 72

3.11.4.4 Zona pellucida removal 73

3.11.4.5 In vitro embryo culture 73

Chapter 4. Construction of an artificial mitochondrial genome

expressing GFP

74

4.1 Introduction 74

4.1.1 Small genome to facilitate mitochondrial internalization and maintenance 75 4.1.2 Green fluorescent protein (gfp) as a mitochondrial reporter gene 76

4.1.3 Mitochondrial complementation 78

4.2 Materials and Methods 79

4.2.1 Oligonucleotides 79

4.2.2 Generation of a mitochondrial gfp gene (mtgfp) 79

4.2.3 Assembly of pmtGFP 80

4.2.4 E. coli transformation and plasmid propagation 81

4.2.5 pmtGFP and pmtBFP sequencing 82

4.2.6 pmtGFP mutagenesis to generate pmtBFP 82

4.2.7 Cell culture 82

4.2.8 Plasmid vector 82

4.2.9 Transfection reagent and preparation of lipoplex 83

4.2.10 Transfection assay 83

4.2.11 Fluorescence in situ hybridization (FISH) 83

4.2.12 Epifluorescence microscopy 84

4.3 Results 84

4.3.1 Generation of the minimitochondrial genome construct pmtGFP 84

4.3.2 Blue fluorescence variant of pmtGFP 91

4.3.3 Expression of pmtGFP following its introduction into cell nuclei 91

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4.4.1 Construction of an artificial mouse mitochondrial genome expressing

GFP 95

4.4.2 The mitochondrial GFP construct 96

4.4.3 The blue fluorescence variant of pmtGFP 98 4.4.4 The minimitochondrial construct pmtGFP is not expressed in the

cytosol 98

4.5 Conclusions 100

Chapter 5. DQAsome as a mitochondrial DNA delivery system

101

5.2.2 Transfection reagents 103

5.2.3 Plasmid vectors 103

5.2.4 Preparation of lipoplex 103

5.2.6 DQAsome cytotoxicity assessments 104

5.2.6.1 Cell growth 104

5.2.6.2 Trypan blue staining 104

5.2.6.3 Mitochondrial JC-1 staining 104 5.2.7 Mitochondrial internalization of pmtGFP DNA 105

5.2.7.1 Fluorescence in situ hybridization (FISH) 105

5.2.7.2 PCR for mtgfp detection 106 5.2.8 Confirmation of mitochondrial GFP expression 106

5.2.8.1 RT-PCR for mtGFP transcription 106

5.2.8.2 Epifluorescence microscopy 106

5.2.8.3 Flow cytometry (FACS Analysis) 107

5.2.8.4 Confocal microscopy 107

5.2.9 Exogenous mtDNA preservation 107

5.2.10 Freezing, thawing and culturing transfected cells 107

5.3 Results 108

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5.3.2.1 Distribution of transfected DNA within cells 113

5.3.2.2 Localization of plasmid DNA in cells transfected with

DQAsome-pmtGFP lipoplex 114

5.3.2.3 Detection of pmtgfp DNA in the mitochondrial fraction of

transfected cells 114

5.3.3 Analysis of mitochondrial GFP expression 116

5.3.3.1 mtgfp transcription using RT-PCR assays 116

5.3.3.2 Time lapse pmtGFP expression post-transfection 116

5.3.3.3 Efficiency of transfection: FACS analysis 120

5.3.3.4 Colocalization of the pmtGFP DNA signal and GFP

expression in transfected cells 121 5.3.4 Mitochondrial preservation of exogenous DNA 122 5.3.5 Persistence of GFP expression after freeze-thawing and culture 123

5.4 Discussion 125

5.4.1 DQAsome cytotoxicity 125

5.4.2 Mitochondrial internalization of pmtGFP 125 5.4.3 Mitochondrial GFP expression observations 127 5.4.4 Mitochondrial preservation of exogenous DNA 129

Chapter 6. Liposome-mediated mitochondrial DNA transfection of

preimplantation embryo

132

6.2.1 Mouse embryos 133

6.2.2 Transfection reagents 133

6.2.2.1 Plasmid vectors 133

6.2.2.2 Liposome and lipoplex 134

6.2.3.1 Nuclear transfection 134

6.2.3.2 Mitochondrial transfection 134 6.2.4 Assessment of transfection efficiency 135

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6.2.4.2 PCR assay for pmtGFP internalization detection 135

6.2.4.3 RT-PCR for mtgfp transcription 136

6.2.4.4 Microscopic observations 136

6.3 Results 137

6.3.1 Nuclear transfection of murine embryonic cells 137

6.3.1.1 Embryo development 137

6.3.1.2 Expression of pDsRed1Mito 137 6.3.2 DQAsome-mediated DNA transfer into mouse embryos 140

6.3.2.1 Embryo development 140

6.3.2.2 Detection of mtgfp by PCR 145

6.3.2.3 Expression of mtgfp 145

6.3.2.4 RT PCR for mtgfp transcription 151

6.4.1 Nuclear transfection at embryonic stage 152 6.4.2 Mitochondrial reconstruction at embryonic stage 153

Chapter 7. Transferring genetically modified mitochondria from one

cell to another: towards the generation of new animal

models

155

7.2.1.1 Mouse embryonic stem cells 156

7.2.1.2 Mouse fetal neuronal stem (mFNS) cells 157 7.2.1.2.1 Generation of mFNS cells 157 7.2.1.2.2 Cryopreservation and thawing of mFNS cells 159

7.2.1.3 Rat fetal fibroblast cells 159

7.2.1.4 Bovine fibroblast cells 159

7.2.1.5 Human kidney 293 cells 160 7.2.2 Transfection of cultured mammalian cells 160 7.2.3 Assessment of transfection efficiency 161

7.2.3.1 Microscopic observations 161

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7.2.4 Nuclear transfer/cytofusion procedures 161

7.2.4.1 Generation of donor cells 161 7.2.4.1.1 Mouse fetal neuronal stem cells generation 161

7.2.4.1.2 Cell sorting 162

7.2.4.1.3 Preparation of cells for micromanipulation and

hand-made cloning (HMC) 162

7.2.4.2 Preparation of recipient cells 163

7.2.4.2.1 Mouse oocytes 163

7.2.4.2.2 Bovine oocytes 163

7.2.4.3 Micromanipulation 164

7.2.4.3.1 Nuclear transfer 164

7.2.4.3.2 Cell injection 165

7.2.4.4 Hand-made cloning (HMC)/cytofusion 165 7.2.4.4.1 Preparation of demi-oocytes 165 7.2.4.4.2 Reconstruction of nuclear transfer embryos 166 7.2.4.4.3 Cytofusion to generate tetraploid embryos 167

7.2.4.4.4 Embryo culture 167

7.2.4.5 Assessment of reconstructed embryos 168

7.2.4.5.1 Embryo development 168

7.2.4.5.2 Epifluorescence observations 168

7.2.4.5.3 PCR analysis 168

7.3 Results 169

7.3.1 Mitochondrial transfection in differentmammalian cell types 169 7.3.2 Transferring GFP positive mFNS cells into eggs/embryos 173

7.3.2.1 pmtGFP transfected mFNS cell as donor cells 173

7.3.2.2 Nuclear transfer embryos generated by micromanipulation 173

7.3.2.3 Eggs/embryos injected with mFNS cells 175

7.3.2.4 Nuclear transfer embryos generated by HMC 178

7.3.2.5 HMC fusion of mFNS cells with oocyte karyoplast 178

7.4.1 Mitochondrial transfection in mammalian cells 181

7.4.1.1 DQAsome mitochondrial transfection 181

7.4.1.2 Mitochondrial expression of mtgfp 182

7.4.1.3 Mitochondrial transfection of pmtGFP in OKO

ES cells 183

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7.4.2.1 The generation of donor cell with modified

mitochondria 184

7.4.2.2 Survival rate and embryo development 185

7.4.2.3 The fate of pmtGFP following cell transfer 187

Chapter 8. General Discussion

191

8.1 A new mitochondrial DNA delivery system 191

8.2 The minimitochondrial genome pmtGFP 192

8.3 Future applications of minimitochondrial genome 193

8.4 Mitochondrial reconstruction in embryos 194

8.5 Concluding remarks 196

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