Effector gene switch-on system by targeted demethylation in human embryonic kidney cells
- 주제(키워드) HEK293 cells , Human cell engineering , Targeted demethylation , Gene switch-on
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 이재성
- 발행년도 2023
- 학위수여년월 2023. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032588
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Human embryonic kidney 293 (HEK293) cells, which have the natural ability to generate human post-translational modifications, are increasingly being used as host cells for biopharmaceutical production. However, the limited production capacity of HEK293 cells requires cell engineering to improve productivity. DNA methylation, which stably regulates gene activity, is a common epigenetic event that occurs via the reversible addition of a methyl group to cytosines in the context of CpG dinucleotides without changing genomic DNA sequences. A programmable CRISPR-Cas9 based demethylase tool consisting of dead Cas9 (dCas9) fused to the catalytic domain of Ten-Eleven Translocation dioxygenase1 (TET1CD) can switch-on specific target genes via targeted demethylation of CpG islands within the promoter region. In this study, we constructed stable HEK293 cell lines expressing TET1CD-dCas9 and effector genes, which were integrated into the well-known human genomic safe harbor, ROSA26 locus, and specific loci silenced by methylation. Using this cell line, we demonstrated that sgRNAs targeting CpG islands of methylated loci can turn-on gene expression. However, the demethylation effect is locus-dependent. We found that targeted demethylation of the RHOXF2B promoter efficiently turned on gene expression by more than 100-fold. Expression was maintained for over a month. By integrating target effector genes, including fluorescent protein, TagBFP, and endoplasmic reticulum-located human protein disulfide isomerase family member, Erp27, into the RHOXF2B 5’UTR region, we constructed RHOXF2B locus-dependent effector gene switch-on system and demonstrated that transgenes can be stably expressed upon demethylation of RHOXF2B promoter triggered by sgRNA expression. Collectively, our data provide a targeted demethylation-based human cell engineering platform that enables sophisticated and stable transgene switch-on for efficient development of human cell lines.
more목차
1. Introduction 1
2. Materials and Methods 4
2.1. Cloning of plasmid 4
2.2. Cell line development (CLD) 5
2.3. Cell culture 6
2.4. Genomic DNA prep and DNA methylation analysis by Bisulfite sequencing 6
2.5. Reverse transcription PCR (RT-PCR) and Quantitative real-time PCR (qRT-PCR) 6
2.6. Western blot analysis 7
2.7. Flow cytometry 8
2.8. 5-aza-2’-deoxycytidine (5-aza-dC) treatment 8
2.9. Live-cell imaging and protein aggregation analysis 8
2.10. Statistical analysis 9
3. Results 18
3.1. Generation of TET1CD-dCas9 stable integrated HEK293 cell lines and functional validation of demethylation-based gene expression 18
3.2. Locus-dependent effect of targeted demethylation on gene expression 22
3.3. Targeted demethylation-based TagBFP fluorescent protein expression at RHOXF2B locus 25
3.4. Transient expression of Erp27 reduces protein aggregation 28
4. Discussion 31
5. References 33
