Article

Recombinant protein expression by targeting pre-selected chromosomal loci

Helmholtz Centre for Infection Research, Braunschweig, Germany.
BMC Biotechnology (Impact Factor: 2.59). 12/2009; 9:100. DOI: 10.1186/1472-6750-9-100
Source: PubMed

ABSTRACT Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs.
We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context.
RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.

0 Followers
 · 
166 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Intraclonal heterogeneity of genetically modified mammalian cells has been observed as a phenomenon that has a strong impact on overall transgene expression levels and that limits the predictability of transgene expression in genetically modified cells, thereby hampering single cell based screening approaches. The underlying mechanism(s) leading to this variance are poorly understood. To study the dynamics and mechanisms of heterogeneity of early stage silencing we analysed the expression in more than 100 independent clones of CHOK1 cells that harbour genetically stable integrates of single copy reporter cassettes driven by EF1α and CMV promoters. Single cell analysis showed intraclonal variability with heterogeneity in expression in genetically uniform populations. DNA methylation is a well known mechanism responsible for silencing of gene expression. Interestingly, loss of expression was not associated with DNA methylation of the CMV promoter. However, in most of the clonal populations expression could be increased by inhibitors of the histone deacetylases (HDACi) suggesting that heterogeneity of transgene expression is crucially governed by histone modifications. Further, to determine if the epigenetic status of the transgene expression is governed by the chromosomal integration locus we targeted heterologous expression cassettes into two chromosomal sites using recombinase mediated cassette exchange (RMCE). The expression status of a particular clone was faithfully re-established when the same promoter used. In this way the problem of early stage cell clone instability can be bypassed. However, upon introduction of an unrelated promoter methylation-independent silencing was observed. Together, these results suggest that histone modifications are the relevant mechanisms by which epigenetic modulation of transgene expression cassettes is governed in the early phase of clone generation. Copyright © 2014. Published by Elsevier B.V.
    Journal of Biotechnology 12/2014; 195. DOI:10.1016/j.jbiotec.2014.12.009 · 2.88 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Gene expression control is critical to increase production of recombinant proteins, fine-tune metabolic pathways and reliably express synthetic pathways. The importance of transcriptional control seems to be most important in eukaryotic systems. In this review, we highlight recent developments in the field of transcriptional engineering with an emphasis on the opportunities and challenges. We discuss the engineering of 'parts' that influence transcriptional throughput including promoters, terminators, and transcription factors as well as the genetic context of the expression cassette. While great strides have been made in the area, the robustness of these parts has been largely untested. This review highlights the importance of considering robustness in biological systems and the limitations that current synthetic parts possess. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Current Opinion in Biotechnology 01/2015; 34C:98-104. DOI:10.1016/j.copbio.2014.12.015 · 8.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The mammalian cell lines HEK293 and CHO have become important expression hosts in structural biology. Generating stable mammalian cell lines remains essential for studying the function and structure of recombinant proteins, despite the emergence of highly efficient transient transfection protocols. Production with stable cell lines can be scaled up easily and high volumetric product yield can be achieved. Protein structure reports of the past two years that used stable cell lines were surveyed for this review. Well-established techniques and novel approaches for generating stable cell lines and stable cell pools are presented, including cell sorting, site-specific recombination, transposons, the Lentivirus system and phage integrases. Host cell line optimization by endoglycosidase overexpression and sequence-specific genome engineering is highlighted. Copyright © 2015. Published by Elsevier Ltd.
    Current Opinion in Structural Biology 03/2015; 32:81-90. DOI:10.1016/j.sbi.2015.03.002 · 8.75 Impact Factor

Full-text (3 Sources)

Download
59 Downloads
Available from
May 29, 2014