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Cited by 13Viral Infectious Diseases and Gene Expression in InsectsGenomics and Chromatin DynamicsVirus-based gene therapy research

The use of a stringent selection system allows the identification of DNA elements that augment gene expression

Open access
Hoeksema, Femke, van Blokland, Rik, Siep, Michel, Hamer, Karien, Siersma, Tjalling, den Blaauwen, Jan, Verhees, John, Otte, Arie P.·2011·Molecular Biotechnology
DOI 10.1007/s12033-010-9344-8PMC PMC3068247
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Abstract

The use of high stringency selection systems often results in the induction of very few recombinant mammalian cell lines, which limits the ability to isolate a cell line with favorable characteristics. The employment of for instance STAR elements in DNA constructs elevates the induced number of colonies and also the protein expression levels in these colonies. Here, we describe a method to systematically identify genomic DNA elements that are able to induce many stably transfected mammalian cell lines. We isolated genomic DNA fragments upstream from the human Rb1 and p73 gene loci and cloned them around an expression cassette that contains a very stringent selection marker. Due to the stringency of the selection marker, hardly any colony survives without flanking DNA elements. We tested fourteen ~3500 bp DNA stretches from the Rb1 and p73 loci. Only two ~3500 bp long DNA fragments, called Rb1E and Rb1F, induced many colonies in the context of the stringent selection system and these colonies displayed high protein expression levels. Functional analysis showed that the Rb1 DNA fragments contained no enhancer, promoter, or STAR activity. Our data show the potential of a methodology to identify novel gene expression augmenting DNA elements in an unbiased manner.

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  • STAR elements were discovered by a genomic screen of elements that would increase transgene expression.

    “They were discovered by a genomic screen of elements that would increase transgene expression.(Kwaks et al., 2003;Sautter and Enenkel, 2005;Hoeksema et al., 2011;Van Blokland et al., 2011;Harraghy et al., 2015).”

    From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Hoeksema et al., 2011

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Cited by

Sorted by citations

Authors
Hoeksema, F., van Blokland, R., Siep, M., Hamer, K., Siersma, T., den Blaauwen, J., Verhees, J., & Otte, A. P.
Affiliations
University of Amsterdam (NL)
Corresponding author
Otte, Arie P.
ORCID
Siersma, Tjalling
Venue
Molecular Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2010-10-29
Volume
48
Issue
1
Pages
19-29
ISSN
  • 1073-6085 (ISSN-L)
  • 1559-0305
DOI
10.1007/s12033-010-9344-8
PMC
PMC3068247
OpenAlex
W2077821394
Cited by
13
References
1
Citations by year
2024: 1 · 2022: 1 · 2021: 3 · 2020: 1 · 2018: 1 · 2017: 3 · 2015: 2 · 2013: 1
FWCI
0.13
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Molecular Biotechnology (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • UvA-DARE (University of Amsterdam) (publishedVersion, other-oa)
  • UvA-DARE (University of Amsterdam) (submittedVersion, other-oa)
  • Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Keywords
protein abundance, selectable marker, enhancer activity, promoter activity
MeSH terms
Animals, Cricetulus, Enzyme-Linked Immunosorbent Assay, Cricetinae, Humans, Transfection, Retinoblastoma Protein, CHO Cells

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