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Engineering Strategies for Intracellular Plasmid Delivery in Mammalian Cells
- Author(s)
- 김동우
- Alternative Author(s)
- Dongwoo Kim
- Advisor
- 이재성
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2022-08
- Language
- eng
- Keyword
- CRISPR/Cas9 system; Gene integration; Mammalian cell engineering; Plasmid delivery; Recombinant protein production
- Alternative Abstract
- Plasmid-based transfection can be utilized in many applications such as plasmid-based gene integration and transient gene expression (TGE)-based therapeutic protein production. These applications preferentially require the translocation of the transfected plasmids into the nucleus. However, the transfected plasmids should overcome intracellular barriers such as DNA degradation and the nuclear envelope. Here, I applied a modified plasmid system and a cell engineering approach to enhance targeted integration and TGE in two mammalian cell lines, Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells. First, a multi-component (MC) system consisting of a single-guide RNA (sgRNA)/double-cut donor (DCD) vector and a Cas9 expression vector was developed to control the ratios of the sgRNA expression cassette and DCD. The MC system increased the knock-in (KI) efficiency of CHO cell lines by approximately 1.5-fold compared to the DCD system by concurrently increasing sgRNA and DCD components relative to Cas9. Second, the Cas9-ZF system was applied to co-localize the CRISPR/Cas9-mediated DNA double-strand breaks and the donor template to increase KI efficiency in CHO cell lines. However, the Cas9-ZF system showed no significant increase in KI efficiency compared to the wild-type Cas9. Third, lysosome engineering was performed in CHO and HEK cell lines to alleviate lysosome-mediated nucleic acid degradation by targeting the RNautophagy/DNautophagy (RDA) pathway. By knocking out LAMP2C and SIDT2, the main players of RDA, we showed that LAMP2C KO effectively improved TGE-based mAb production by up to 2.82-fold increase in specific mAb productivity. This study demonstrated the importance of translocating transfected plasmids into the nucleus and provided a novel plasmid system and cell lines for the effective delivery of plasmids and relevant components for gene integration and expression.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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