Title Page
Abstract
Contents
1. Introduction 11
2. Materials and methods 14
2.1. Cloning of plasmid 14
2.2. Cell line development (CLD) 15
2.3. Cell culture 15
2.4. Genomic DNA prep and DNA methylation analysis by Bisulfite sequencing 16
2.5. Reverse transcription PCR (RT-PCR) and Quantitative real-time PCR (qRT-PCR) 16
2.6. Western blot analysis 17
2.7. Flow cytometry 18
2.8. 5-aza-2'-deoxycytidine (5-aza-dC) treatment 18
2.9. Live-cell imaging and protein aggregation analysis 18
2.10. Statistical analysis. 19
3. Results 28
3.1. Generation of TET1CD-dCas9 stable integrated HEK293 cell lines and functional validation of demethylation-based gene expression 28
3.2. Locus-dependent effect of targeted demethylation on gene expression 32
3.3. Targeted demethylation-based TagBFP fluorescent protein expression at RHOXF2B locus 35
3.4. Transient expression of Erp27 reduces protein aggregation 38
4. Discussion 41
References 43
Table 1. Plasmids used in this study 20
Table 2. Primer sequences used in this study 22
Figure. 1. Construction of TET1CD-dCas9 stable integrated cell line (TRC). (A) Schematic overview of stable integration of TET1CD-dCas9 gene at the ROSA26... 31
Figure. 2. Locus-dependent effect of targeted demethylation on gene expression. (A) RT-PCR of target genes (GSX2, FOXL1) after transfection with gRNA expression... 34
Figure. 3. Construction of TagBFP expression switch at the RHOXF2/2B locus. (A) Schematic overview of transgene switch-on system at RHOXF2/2B locus. (B)... 37
Figure. 4. Effect of Erp27 transient expression on reducing protein aggregation (A) RT-PCR results of Erp27 in the presence of various concentrations of 5-aza-dC (0,... 40