AJOU Central Library Repository: Application of Endogenous p21 Dependent Transgene Switch in CHO Cell Engineering

Application of Endogenous p21 Dependent Transgene Switch in CHO Cell Engineering

Keyword: Chinese hamster ovary cells; Endogenous gene tagging; Targeted integration; Transgene expression regulation

Alternative Abstract: Numerous engineering efforts have been made in Chinese hamster ovary (CHO) cells for high level production of therapeutic proteins and overall improvement in cellular properties. However, the dynamic regulation of transgene expression is limited in current systems due to randomly integrated transgenes and limited information on the regulatory elements in CHO cells. In this study, we investigated the effective regulation of transgene expression via targeted integration-based endogenous gene tagging with engineering target genes. Targeted integration of EGFP-human Bcl-2, which encodes an anti-apoptosis effector protein, into the endogenous CHO p21CDKN1A locus effectively reduced the apoptosis in transfected pools of cells and clonal populations, compared with random populations in which human Bcl-2 expression was driven by CMV promoter. Endogenous p21 and EGFP-human Bcl-2 displayed similar expression dynamics in batch cultures, and the anti-apoptotic effect altered the expression pattern of endogenous p21 showing the mutual influences between expression of p21 and Bcl-2. Furthermore, we demonstrated the inducible expression of transgenes by adding low concentrations of hydroxyurea. The present engineering strategy will provide a valuable CHO cell engineering tool that can be used to control dynamic transgene expression in accordance with different cellular states.

Appears in Collections:: Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)

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