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Cited by 122CRISPR and Genetic EngineeringVirus-based gene therapy researchRNA Interference and Gene Delivery

An improved platform for functional assessment of large protein libraries in mammalian cells

Open access
Matreyek, Kenneth A, Stephany, Jason J, Chiasson, Melissa A, Hasle, Nicholas, Fowler, Douglas M·2019·Nucleic Acids Research
DOI 10.1093/nar/gkz910PMC PMC7145622
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Abstract

Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.

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  • HEK293 cells have been modified to optimise variant library integration, rendering them particularly useful for generic functional assays such as VAMP-seq, which determines variant effects on protein abundance.

    “HEK293 cells have moreover been modified to optimise variant library integration, rendering them particularly useful for generic functional assays such as VAMP-seq, which determines variant effects on protein abundance (70, 71).”

    From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [71]

    The Introduction and Results (‘Lentiviral landing pads expedite cell engineering’; page unavailable) describe improved landing pads in HEK 293T cells that increase recombination and enable variant-library expression for multiplex assays. Our copy does not identify VAMP-seq as an application or describe its protein-abundance readout, so that part of the sentence is not supported by this source. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

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Cited by

Sorted by citations

Authors
Matreyek, K. A., Stephany, J. J., Chiasson, M. A., Hasle, N., & Fowler, D. M.
Affiliations
University of Washington (US)
Corresponding author
  • Matreyek, Kenneth A
  • Fowler, Douglas M
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2019-10-15
Volume
48
Issue
1
Pages
e1-e1
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkz910
PMC
PMC7145622
OpenAlex
W2980355535
Cited by
122
References
3
Citations by year
2026: 16 · 2025: 23 · 2024: 34 · 2023: 23 · 2022: 6 · 2021: 16 · 2020: 4
FWCI
2.13
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • National Institute of General Medical Sciences (R01GM109110)
  • National Human Genome Research Institute (RM1HG010461)
  • American Cancer Society Fellowship (PF-15-221-01)
  • National Cancer Institute Interdisciplinary Training (T32CA080416)
  • CIFAR Azrieli
  • National Science Foundation Graduate Research
  • National Cancer Institute (F30CA236335)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
lentiviral vectors, oncogenes, tumor suppressor, cell growth, mammalian cell lines
MeSH terms
Red Fluorescent Protein, Animals, Biological Assay, Genetic Vectors, Humans, Luminescent Proteins, Oncogenes, Plasmids, Recombinant Proteins, Recombination, Genetic, Gene Library, Lentivirus, HT29 Cells, Transgenes, Tumor Suppressor Proteins, NIH 3T3 Cells, Recombinases, Green Fluorescent Proteins, Mice, HEK293 Cells

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