Differences in developmental competence, gene expression and effects of altering epigenetic ...

157 Pages·2016·6.64 MB·English

Differences in developmental competence, gene
expression and effects of altering epigenetic status
between male and female cloned buffalo embryos
produced through hand-guided cloning

THESIS SUBMITTED TO THE
NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY
IN
ANIMAL BIOTECHNOLOGY

BY
ANJIT SANDHU
M.Sc. (Biotechnology)

ANIMAL BIOTECHNOLOGY CENTRE
NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY)
KARNAL-132 001 (HARYANA), INDIA

2014
Regn. No. 1121003

Dedicated
to
My parents, Brothers
&
My loving Husband

CONTENTS
Chapter Title Page
No.
1.0 INTRODUCTION 1-5
2.0 REVIEW OF LITERATURE 6-31
2.1 Somatic cell nuclear transfer or animal cloning 6
2.2 Historical overview 7
2.3 Status of cloning in buffalo 9
2.4 Factors contributing the efficiency of cloning 12
2.4.1 Recipient oocyte 13
2.4.2. Donor cell type 14
2.4.3 Effect of donor age 14
2.4.4 Donor cell-cycle stage 15
2.4.5 Epigenetic modifications 15
2.5 Nuclear reprogramming after SCNT 15
2.6 Epigenetics 17
2.6.1 DNA methylation 18
2.6.2 Histone modifications 18
2.7 Epigenetic Reprogramming following SCNT 19
2.8 X-Chromosome Inactivation 21
2.8.1 Mechanism of X-chromosome Inactivation 22
2.8.1.1 The Xist gene 23
2.8.1.2 Inhibitors of X chromosome inactivation 26
2.8.2 X chromosome Inactivation in pre-implantation 26
embryos
2.8.3 Problems in female cloned embryo development 27
2.8.4 Effect of Epigenetic modifiers on X-chromosome 31
Inactivation
3.0 MATERIALS AND METHODS 32-58
3.1 Materials 32
3.1.1 Plasticware and Glassware 32
3.1.2 Chemicals, cell culture media and supplements 32
3.1.3 Equipments 33
3.1.3.1 Microscopes 33
3.1.3.2 CO incubator 34
2
3.1.3.3 Laminar flow cabinet 34
Chapter Title Page
No.
3.1.3.4 Centrifuge 35
3.1.3.5 Vortex shaker 35
3.1.3.6 Electrofusion equipment 35
3.1.3.7 ELISA reader 35
3.1.3.8 Thermal cycler 35
3.1.3.9 Real time PCR 36
3.1.3.10 Electrophoresis unit and gel 36
documentation
3.1.4 Biologicals 36
3.1.4.1 Ovaries and oocytes 36
3.1.4.2 Buffalo follicular fluid 36
3.1.4.3 Tissues for Somatic Cell Cultures 37
3.2 Methods 37
3.2.1 Preparation of different media 37
3.2.2 Somatic cell cultures 37
3.2.2.1 Establishment of fibroblast primary cell cultures 37
3.2.2.2 Sub-culturing the cells 38
3.2.2.3 Immunocytochemical characterization of skin-derived 38
somatic cells
3.2.2.4 Maintenance of somatic cell cultures 39
3.2.2.5 Cryopreservation by slow freezing 39
3.2.2.6 BrdU cell proliferation assay 40
3.2.2.7 TSA and 5-aza-dC treatment to donor cells 40
3.2.3 In vitro embryo production 41
3.2.3.1 Handmade cloning 41
3.2.3.1.1 Collection and classification of oocytes 41
3.2.3.1.2 In vitro maturation of oocytes 42
3.2.3.1.3 Processing of IVM oocytes for 42
preparation of cytoplasts
3.2.3.1.3.1 Denuding the oocytes 42
3.2.3.1.3.2 Removal of zona pellucid 42
3.2.3.1.3.3 Protrusion cone-guided 43
bisection and Hoechst
33342 selection
3.2.3.1.4 Preparation of donor cells for nuclear 43
transfer
Chapter Title Page
No.
3.2.3.1.5 Pairing and electrofusion of donor cells 43
and cytoplasts
3.2.3.1.5.1 Single step fusion method 44
3.2.3.1.6 Activation of reconstructed oocytes 44
3.2.3.1.7 In Vitro culture of embryos 44
3.2.3.1.8 Cryopreservation of embryos 45
3.2.3.1.9 Synchronization of recipients 45
3.2.3.1.10 Loading of embryos in French straws 46
3.2.3.1.11 Embryo transfer to recipients 46
3.2.3.1.12 Monitoring and Diagnosis of 46
Pregnancies
3.2.3.2 In Vitro Fertilization 46
3.2.4 Assessment of embryo development apoptosis by TUNEL assay 47
3.2.5 Immunofluorescence staining for epigenetic markers in cloned 48
blastocysts
3.2.6 Quantitative expression of genes 49
3.2.6.1 RNase-free plasticware 49
3.2.6.2 Designing of primers 50
3.2.6.3 Extraction of total RNA from somatic cells and cloned 50
embryos
3.2.6.3.1 RNA isolation protocol 52
3.2.6.3.1.1 Trizol method for Somatic 52
cells
3.2.6.3.1.2 RNA isolation from cloned 53
blastocyst
3.2.6.4 cDNA preparation 54
3.2.6.5 Comparative quantification of mRNA in donor cells 55
and cloned embryos using Real-time PCR
3.3 Experimental designs 57
3.3.1 Comparison of the developmental competence, 57
quality and epigenetic status between male and
female cloned embryos
3.3.2 Comparison of the expression level of genes between 57
male and female embryos.
3.3.3 Effects of treatment with epigenetic modifiers on the 58
developmental competence and quantitative
expression of above mentioned genes in male and
female cloned embryos
58
3.4 Statistical analysis
Chapter Title Page
No.
4 RESULTS AND DISCUSSION 59-77
4.1 Comparison of the developmental competence, quality and 59
epigenetic status between male and female cloned embryos
4.1.1 Establishment and characterization of cell lines 59
4.1.2 Production of cloned male and female embryos and 59
comparison of their developmental competence and
quality
4.1.3 Evaluation of the epigenetic status of male and 61
female embryos
4.2 Comparison of the expression level of genes between male and 63
female embryos
4.2.1 Measurement of quantitative expression of genes by 63
qPCR
4.3 Effects of treatment with epigenetic modifiers on the 63
developmental competence and quantitative expression of
above mentioned genes between male and female cloned
embryos
4.3.1 Evaluation of the effects of treatment with epigenetic 63
modifiers on the developmental competence and
quality of male and female cloned embryos
4.3.2 Evaluation of the epigenetic status of epigenetic 66
modifier-treated male and female embryos
4.3.3 Measurement of quantitative expression of genes by 67
qPCR in epigenetic modifier-treated male and
female embryos
5.0 SUMMARY AND CONCLUSIONS 78-86
BIBLIOGRAPHY i-xxiii
ANNEXURE xxiv-
xxx