List of abbreviations and measurement units
Table of contents
List of abbreviations and measurement units
i
Table of contents
iii
List of Figures
xii
List of Tables
xv
List of Publications
xvi
Abstract
xvii
Declaration
xix
Acknowledgements
xx
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
2.2 Human mitochondrial diseases
12
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
2.4.2.3.1 Cis-acting elements
32
iii
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
Chapter 3. General Materials and Methods
46
47
47
3.1 DNA
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
3.3 Transfection
56
iv
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
3.11.1 Mice
71
71
v
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
4.1 Introduction
74
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
4.4 Discussion
95
vi
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
Chapter 5. DQAsome as a mitochondrial DNA delivery system
100
101
5.1 Introduction
101
5.2 Materials and Methods
102
5.2.1 Cell culture
102
5.2.2 Transfection reagents
103
5.2.3 Plasmid vectors
103
5.2.4 Preparation of lipoplex
103
5.2.5 Transfection assays
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
5.3.1 DQAsome is cytotoxic at high concentrations
108
5.3.2 Mitochondrial internalization of pmtGFP
113
vii
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
5.5 Conclusions
130
Chapter 6. Liposome-mediated mitochondrial DNA transfection of
preimplantation embryo
132
6.1 Introduction
132
6.2 Materials and Methods
133
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 Transfection assays
134
6.2.3.1 Nuclear transfection
134
6.2.3.2 Mitochondrial transfection
134
6.2.4 Assessment of transfection efficiency
6.2.4.1 Preparation of embryo lysates for PCR analysis
135
135
viii
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
137
6.3 Results
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 Discussion
152
6.4.1 Nuclear transfection at embryonic stage
152
6.4.2 Mitochondrial reconstruction at embryonic stage
153
154
6.5 Conclusions
Chapter 7. Transferring genetically modified mitochondria from one
cell to another: towards the generation of new animal
models
155
7.1 Introduction
155
7.2 Materials and Methods
156
7.2.1 Cell culture
156
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
7.2.1.2.2 Cryopreservation and thawing of mFNS cells
157
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
7.2.3.2 Flow cytometry analysis
161
ix
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 handmade cloning (HMC)
162
7.2.4.2 Preparation of recipient cells
7.2.4.2.1 Mouse oocytes
7.2.4.2.2 Bovine oocytes
7.2.4.3 Micromanipulation
7.2.4.3.1 Nuclear transfer
7.2.4.3.2 Cell injection
7.2.4.4 Hand-made cloning (HMC)/cytofusion
7.2.4.4.1 Preparation of demi-oocytes
7.2.4.4.2 Reconstruction of nuclear transfer embryos
7.2.4.4.3 Cytofusion to generate tetraploid embryos
7.2.4.4.4 Embryo culture
7.2.4.5 Assessment of reconstructed embryos
7.2.4.5.1 Embryo development
7.2.4.5.2 Epifluorescence observations
7.2.4.5.3 PCR analysis
7.3 Results
163
163
163
164
164
165
165
165
166
167
167
168
168
168
168
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 Discussion
181
7.4.1 Mitochondrial transfection in mammalian cells
181
7.4.1.1 DQAsome mitochondrial transfection
7.4.1.2 Mitochondrial expression of mtgfp
7.4.1.3 Mitochondrial transfection of pmtGFP in OKO
ES cells
7.4.2 Embryos carrying reconstructed mitochondria
181
182
183
184
x
7.4.2.1 The generation of donor cell with modified
mitochondria
7.4.2.2 Survival rate and embryo development
7.4.2.3 The fate of pmtGFP following cell transfer
7.5 Conclusions
Chapter 8. General Discussion
184
185
187
190
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
References
197
xi
xii
List of abbreviations and measurement units
i
Table of contents
iii
List of Figures
xii
List of Tables
xv
List of Publications
xvi
Abstract
xvii
Declaration
xix
Acknowledgements
xx
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
2.2 Human mitochondrial diseases
12
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
2.4.2.3.1 Cis-acting elements
32
iii
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
Chapter 3. General Materials and Methods
46
47
47
3.1 DNA
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
3.3 Transfection
56
iv
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
3.11.1 Mice
71
71
v
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
4.1 Introduction
74
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
4.4 Discussion
95
vi
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
Chapter 5. DQAsome as a mitochondrial DNA delivery system
100
101
5.1 Introduction
101
5.2 Materials and Methods
102
5.2.1 Cell culture
102
5.2.2 Transfection reagents
103
5.2.3 Plasmid vectors
103
5.2.4 Preparation of lipoplex
103
5.2.5 Transfection assays
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
5.3.1 DQAsome is cytotoxic at high concentrations
108
5.3.2 Mitochondrial internalization of pmtGFP
113
vii
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
5.5 Conclusions
130
Chapter 6. Liposome-mediated mitochondrial DNA transfection of
preimplantation embryo
132
6.1 Introduction
132
6.2 Materials and Methods
133
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 Transfection assays
134
6.2.3.1 Nuclear transfection
134
6.2.3.2 Mitochondrial transfection
134
6.2.4 Assessment of transfection efficiency
6.2.4.1 Preparation of embryo lysates for PCR analysis
135
135
viii
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
137
6.3 Results
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 Discussion
152
6.4.1 Nuclear transfection at embryonic stage
152
6.4.2 Mitochondrial reconstruction at embryonic stage
153
154
6.5 Conclusions
Chapter 7. Transferring genetically modified mitochondria from one
cell to another: towards the generation of new animal
models
155
7.1 Introduction
155
7.2 Materials and Methods
156
7.2.1 Cell culture
156
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
7.2.1.2.2 Cryopreservation and thawing of mFNS cells
157
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
7.2.3.2 Flow cytometry analysis
161
ix
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 handmade cloning (HMC)
162
7.2.4.2 Preparation of recipient cells
7.2.4.2.1 Mouse oocytes
7.2.4.2.2 Bovine oocytes
7.2.4.3 Micromanipulation
7.2.4.3.1 Nuclear transfer
7.2.4.3.2 Cell injection
7.2.4.4 Hand-made cloning (HMC)/cytofusion
7.2.4.4.1 Preparation of demi-oocytes
7.2.4.4.2 Reconstruction of nuclear transfer embryos
7.2.4.4.3 Cytofusion to generate tetraploid embryos
7.2.4.4.4 Embryo culture
7.2.4.5 Assessment of reconstructed embryos
7.2.4.5.1 Embryo development
7.2.4.5.2 Epifluorescence observations
7.2.4.5.3 PCR analysis
7.3 Results
163
163
163
164
164
165
165
165
166
167
167
168
168
168
168
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 Discussion
181
7.4.1 Mitochondrial transfection in mammalian cells
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7.4.1.1 DQAsome mitochondrial transfection
7.4.1.2 Mitochondrial expression of mtgfp
7.4.1.3 Mitochondrial transfection of pmtGFP in OKO
ES cells
7.4.2 Embryos carrying reconstructed mitochondria
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7.4.2.1 The generation of donor cell with modified
mitochondria
7.4.2.2 Survival rate and embryo development
7.4.2.3 The fate of pmtGFP following cell transfer
7.5 Conclusions
Chapter 8. General Discussion
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8.1 A new mitochondrial DNA delivery system
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8.2 The minimitochondrial genome pmtGFP
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8.3 Future applications of minimitochondrial genome
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8.4 Mitochondrial reconstruction in embryos
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8.5 Concluding remarks
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References
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