표제지
ACKNOWLEDGEMENT
목차
ABBREVIATIONS 11
ABSTRACT 12
1. INTRODUCTION 14
2. MATERIALS AND METHODS 18
2.1 Isolation of ICM from the mouse blastocyst and establishment of ES cell lines 18
2.2 Isolation of PGCs from the developing gonads and establishment of EG cell lines 19
2.3 Mitotic inactivation of mEF cells 20
2.4 Characterization of B6 mouse ES and EG cells 20
2.4.1 Alkaline phosphatase staining 20
2.4.2 Immnocytochemical methods 21
2.4.3 In situ hybridization 22
2.4.4 Chromosomal analysis 23
2.4.5 Spontaneous differentiation 23
2.4.6 RT-PCR analysis of Oct-4 and Nanog genes 23
2.4.7 Telomerase activity (TRAP assay) 24
2.5. Differentiation into neural tissues 25
2.5.1 Transfection of EGFP-N3 vector using Exgen 500 25
2.5.2 Induction of neural differentiation of mouse ES cells 26
2.5.3 RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) analysis 26
2.5.4 Immunocytochemical methods 27
2.6. Transplantation of B6 mouse ES cells 27
2.6.1 Detection of major viruses to be used in cell transplantation studies 27
2.6.2 Procedures for cell preparation and transplantation 28
2.6.3 Analysis of the grafted brain cryosections 28
2.6.4 Teratoma formation in SCID mice 29
3. RESULTS 30
3.1. Characterization of B6 mouse ES and mEG cells 30
3.1.1 Derivation of B6 mouse ES cell lines 30
3.1.2 Derivation of B6 mouse EG cell lines 30
3.1.3 Marker expression and karyotying analysis of B6 mouse ES and EG cells 31
3.2. Differentiation into neural tissues 33
3.2.1 Neurite formation from mouse B5 and B6 ES lines
using PA6 conditioned medium 33
3.2.2 Expression of pluripotent stem cells markers, Oct-4, Nanog, Cripto, and ERas genes in the differentiation of B6 mouse ES cells, analyzed by RT-PCR 34
3.2.3 Differentiation into neural lineages by immnocytochemical study 35
3.2.4 Differentiation into neural lineages by RT-PCR analysis 37
3.3. In vivo application of B6 mouse ES cells 39
3.3.1 Detection of viral contamination in ES cells 39
3.3.2 Survival and overall distribution of the grafted GFP-positive ES cells 39
3.3.3 Teratoma formation in SCID mice 42
4. DISCUSSION 43
REFERENCES 47
SUMMARY 56
Table 1. Antibodies used in the immunocytochemical study 36
Table 2. List of Primer sequences used in the RT-PCRs 38
Table 3. List of the major viruses analyzed in this study 40
Fig.1 Morphology of undifferentiated ES and EG cell colonies 9
Fig.2 Spontaneous differentiation of B6 mouse ES and EG cells in
suspension culture 9
Fig.3 Marker expression of mouse ES and EG cell lines derived from
C57BL/6 mice 9
Fig.4 Karyotyping analysis on the B6 mouse ES and EG cell lines 9
Fig.5 Expression of Oct-4 gene in B6 mouse ES and mEG cell lines,
detected by RT-PCR 9
Fig.6 Expression of Nanog gene in B6 mouse ES and mEG cell lines,
Fig.7 Telomerase assay on mouse B6 ES and EG cells lines using
TRAP assay 9
Fig.8 Neurite formation from mouse B5 and B6 ES cell lines using
PA6 conditioned medium 9
Fig.9 Comparison of neurite formation from various mouse ES and EG
cell lines using PA6 conditioned medium 9
Fig.10 Expression of Oct-4 and Nanog genes in various stages of ES
cell differentiation, detected by RT-PCR 10
Fig.11 Confocal analysis of neuronal differentiation from B5 mouse ES
cells using PA6 Conditioned medium 10
Fig.12 Confocal analysis of neuronal differentiation from B6 mouse ES
cells using PA6 conditioned medium 10
Fig.13 Expression of neuronal markers from various stages of
differentiation in B6 mouse ES cells, analyzed by RT-PCR 10
Fig.14 Expression of pluripotent stem cells markers, Oct-4, Cripto and
Eras genes in the differentiation of B6 mouse ES cells, analyzed
by RT-PCR 10
Fig.15 Genomic PCR and RT-PCR analyses to detect viral contamination
in both ES and EG cells 10
Fig.16 Detection of EGFP-labelled B6 mouse ES cells following
transplantation into the adult striatum and the newborn mouse
brain 10
Fig.17 Histological analysis of teratomas formed by injection of B6
mouse ES cells 10