Title Page
Contents
국문 초록 8
ABSTRACT 9
Ⅰ. Introduction 10
Ⅱ. Materials and Methods 13
1. Cell culture 13
2. Generation of αMHC-EGFP KI reporter cell line 13
3. Differentiation into three germ layers 15
4. Differentiation into cardiomyocytes 15
5. Realtime-PCR (qPCR) analysis 15
6. Immunocytochemistry 17
7. Flow cytometry 19
8. Karyotyping, short term repeat (STR) analysis, and mycoplasma detection 19
9. Plasmid random integration assay and off-target sequencing 20
10. Ethic/GMO work approvals 22
Ⅲ. Results 23
1. Verification of insertion of αMHC-EGFP gene using CRISPR/Cas9 and genetic abnormality 23
2. Characterization of knock-in αMHC-EGFP hPSC line 28
3. Differentiation into Cardiomyocyte and validation of fidelity of αMHC-EGFP hPSC line 30
Ⅳ. Discussion 32
Ⅴ. References 36
Table 1. Primers for verification of knock-in construct 14
Table 2. Primers for realtime-PCR (qPCR) 16
Table 3. Antibodies for immunocytochemistry 18
Table 4. Primers for random integration and off-target 21
Figure 1. Verification of αMHC-EGFP targeting and donor construct. 25
Figure 2. Analysis of αMHC-EGFP clone genomic abnormality and the presence of mycoplasma contamination. 26
Figure 3. Analysis of genetic abnormality caused by side effect of CRISPR/Cas9. 27
Figure 4. Characterization of αMHC-EGFP clone compared to hESCs. 29
Figure 5. Validation of fidelity of αMHC-EGFP hPSC line through differentiation into cardiomyocytes. 31