Effects of 3′-untranslated region modifications on transgene expression in the human embryonic kidney 293 cell line
- 주제(키워드) 3'UTR engineering , mRNA stability , transgene expression , stable cell line , HEK293 cell
- 주제(DDC) 547
- 발행기관 아주대학교 일반대학원
- 지도교수 Jae Seong Lee
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000033288
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Improving the productivity of recombinant cell lines is a key goal of mammalian cell line development (CLD), which can be achieved via different strategies, such as the control of mRNA degradation. However, such strategies have received little attention. Moreover, only a few studies have attempted to use the 3′-untranslated region (UTR) to alter the mRNA stability and protein expression levels in CLD. Whether this strategy can help in controlling the transgene expression remains unclear. Therefore, to determine the utility of 3′-UTR engineering for CLD, we aimed to assess whether mRNA stability and transgene expression can be altered by inserting additional 3′- UTR sequences into the commonly used expression cassette in the representative human cell line, human embryonic kidney (HEK) 293. We designed synthetic 3′- UTRs composed of various motifs derived from endogenous genes based on global mRNA stability and transcriptome data of HEK293 cells. Although changes in the mRNA half-life did not have any direct effects, the inserted 3′-UTR sequences exhibited potential to modulate the mRNA half-life and protein expression levels. These results demonstrate the potential use of 3′-UTR engineering for the fine-tuning of transgene expression and tight regulation of gene of interest in mammalian cell line engineering.
more목차
1. Introduction 1
2. Materials and Methods 3
2.1. Plasmid construction 3
2.2. Cell culture and generation of stable cell line 3
2.3. Flow cytometry 4
2.4. Genomic DNA, RNA, and cDNA preparation 4
2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) 5
2.6. Measurement of stable expression level 5
2.7. Copy number validation 5
2.8. Measurement of basal transcript level 5
2.9. Measurement of mRNA half-life 6
2.10. Gene ontology (GO) analysis 7
2.11. Statistical analysis 7
3. Results 15
3.1. Generation of human embryonic kidney 293 (HEK293) stable cell lines with additional known 3' untranslated regions (3'UTR) and their effects on transgene expression levels 15
3.2. Investigation of synthetic 3'UTR sequences expected to be associated with mRNA stability 20
3.3. Evaluation of mRNA stability and changes in transgene expression level by synthetic 3'UTRs 25
3.4. Evaluation of the impact of promoter strength on transgene expression 28
4. Discussion 31
5. References 34
