Systematic Evaluation of Site-Specific Recombinant Gene Expression for Programmable Mammalian Cell Engineering

Nuša Pristovšek, Saranya Nallapareddy, Lise Marie Grav, Hooman Hefzi, Nathan E. Lewis, Peter Rugbjerg, Henning Gram Hansen, Gyun Min Lee*, Mikael Rørdam Andersen, Helene Faustrup Kildegaard

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review


Many 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.

Original languageEnglish
JournalACS Synthetic Biology
Issue number4
Pages (from-to)758-774
Number of pages17
Publication statusPublished - 2019


  • Chinese hamster ovary cells
  • Gene expression
  • Mammalian cell engineering
  • Site-specific integration
  • Synthetic biology


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