Title Page
Contents
국문초록 8
ABSTRACT 9
Ⅰ. Introduction 10
Ⅱ. Materials and Methods 14
1. Human PSC culture and TUBB3-mCherry KI reporter cell line generation 14
2. In vitro differentiation into three germ layers and neuronal differentiation 15
3. Immunocytochemistry 16
4. RNA extraction, cDNA synthesis, and realtime-PCR (qPCR) analysis 18
5. Off-target analysis 18
6. Mycoplasma testing 20
7. Karyotyping and short tandem repeat (STR) analysis 20
8. Ethical/GMO work approvals 20
Ⅲ. Result 21
1. Generation of TUBB3-mCherry KI reporter cell line 21
2. Off-targets and backbone confirmation in TUBB3-mCherry KI cells 24
3. TUBB3-mCherry KI reporter cell line retained pluripotency 27
4. mCherry expression helps to monitor neuronal differentiation 29
Ⅳ. Discussion 31
Ⅴ. References 34
Table 1. Antibodies and stains used for immunocytochemistry/flow-cytometry 17
Table 2. Primers and Oligonucleotides used in this study 19
Figure 1. Schematic overview depicting the targeting strategy for a donor construct and sgRNA sequence to target a region of stop codon... 22
Figure 2. The TUBB3-mCherry KI reporter cell line was identified through genomic DNA PCR and sequencing 23
Figure 3. There was no insertion of donor backbone and off-target on the TUBB3-mCherry KI reporter cell line 25
Figure 4. STR analysis or other genotypic identity evidence types 26
Figure 5. The pluripotency of the TUBB3-mCherry reporter cells were identified 28
Figure 6. TUBB3-mCherry reporter cells expressed mCherry during neuronal differentiation 30