Journal article
Systematic Evaluation of Site-Specific Recombinant Gene Expression for Programmable Mammalian Cell Engineering
CHO Cell Line Engineering and Design, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
Korea Advanced Institute of Science and Technology2
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
University of California at San Diego4
Bacterial Synthetic Biology, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark5
Afdelingen for Produktionsudvikling, Center for Bachelor of Engineering Studies, Technical University of Denmark6
Center for Bachelor of Engineering Studies, Technical University of Denmark7
Section for Synthetic Biology, Department of Biotechnology and Biomedicine, Technical University of Denmark8
Network Engineering of Eukaryotic Cell factories, Section for Synthetic Biology, Department of Biotechnology and Biomedicine, Technical University of Denmark9
Department of Biotechnology and Biomedicine, Technical University of Denmark10
...and 0 moreMany branches of biology depend on stable and predictable recombinant gene expression, which has been achieved in recent years through targeted integration of the recombinant gene into defined integration sites. However, transcriptional levels of recombinant genes in characterized integration sites are controlled by multiple components of the integrated expression cassette.
Lack of readily available tools has inhibited meaningful experimental investigation of the interplay between the integration site and the expression cassette components. Here we show in a systematic manner how multiple components contribute to final net expression of recombinant genes in a characterized integration site.
We develop a CRISPR/Cas9-based toolbox for construction of mammalian cell lines with targeted integration of a landing pad, containing a recombinant gene under defined 5′ proximal regulatory elements. Generated site-specific recombinant cell lines can be used in a streamlined recombinase-mediated cassette exchange for fast screening of different expression cassettes.
Using the developed toolbox, we show that different 5′ proximal regulatory elements generate distinct and robust recombinant gene expression patterns in defined integration sites of CHO cells with a wide range of transcriptional outputs. This approach facilitates the generation of user-defined and product-specific gene expression patterns for programmable mammalian cell engineering.
Language: | English |
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Publisher: | American Chemical Society |
Year: | 2019 |
Pages: | 758-774 |
ISSN: | 21615063 |
Types: | Journal article |
DOI: | 10.1021/acssynbio.8b00453 |
ORCIDs: | Pristovšek, Nuša , Rugbjerg, Peter , Kildegaard, Helene Faustrup , Grav, Lise Marie and Andersen, Mikael Rørdam |
CRISPR Chinese hamster ovary cells Gene expression Mammalian cell engineering Site-specific integration Synthetic biology
Animals CHO Cells CRISPR-Cas Systems Cell Engineering Cell Line Chinese hamsterovary cells Clustered Regularly Interspaced Short Palindromic Repeats Cricetulus Gene Expression Mammals Recombinant Proteins Recombinases Regulatory Sequences, Nucleic Acid Transcription, Genetic gene expression mammalian cell engineering site-specific integration synthetic biology