Dataset Information

Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals.

ABSTRACT: Interleukin-7 (IL-7) is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP) gene, drug selection cassette, and modified homologous arms which are considered to enhance the translation of the eGFP reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the IL-7 start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report IL-7 gene transcription based on the eGFP protein signal. Furthermore, we utilized the cells to run a pilot screen campaign for IL-7-upregulating chemicals in a high-throughput format, and identified a chemical that can up-regulate IL-7 gene transcription. Collectively, these results suggest that our IL-7 reporter system can be utilized in large-scale chemical library screening to reveal novel IL-7 regulatory pathways and to identify potential drugs for development of new treatments in immunodeficiency disease.

SUBMITTER: Cho YS

PROVIDER: S-EPMC5010243 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals.

Cho Yeon Sook YS Kim Byung Soo BS Sim Chan Kyu CK Kim Inki I Lee Myeong Sup MS

PloS one 20160902 9

Interleukin-7 (IL-7) is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP) gen ...[more]

PMID: 27589392

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Project description:BackgroundEstablishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES) cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12) p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells.ResultsA series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i) a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii) the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii) the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv) expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v) utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi) demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii) germline transmission of the IL-12 p40 reporter gene.ConclusionIL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using a small aliquot of the culture medium. Remarkably, expression of Il-12 p40 does not affect the pluripotency of mouse ES cells. To our knowledge, human IL-12 p40 is the first secreted reporter protein suitable for high-throughput screening of mouse ES cells. In comparison to other secreted reporters, such as the widely used alkaline phosphatase (SEAP) reporter, the IL-12 p40 reporter system offers other real advantages.

Project description:The ubiquitous presence of more than 80,000 chemicals in thousands of consumer products used on a daily basis stresses the need for screening a broader set of chemicals than the traditional well-studied suspect chemicals. This high-throughput screening combines stochastic chemical-product usage with mass balance-based exposure models and toxicity data to prioritize risks associated with household products. We first characterize product usage using the stochastic SHEDS-HT model and chemical content in common household products from the CPDat database, the chemical amounts applied daily varying over more than six orders of magnitude, from mg to kg. We then estimate multi-pathways near- and far-field exposures for 5,500 chemical-product combinations, applying an extended USEtox model to calculate product intake fractions ranging from 0.001 to ∼1, and exposure doses varying over more than nine orders of magnitude. Combining exposure doses with chemical-specific dose-responses and reference doses shows that risks can be substantial for multiple home maintenance products, such as paints or paint strippers, for some home-applied pesticides, leave-on personal care products, and cleaning products. Sixty percent of the chemical-product combinations have hazard quotients exceeding 1, and 9% of the combinations have lifetime cancer risks exceeding 10-4 . Population-level impacts of household products ingredients can be substantial, representing 5 to 100 minutes of healthy life lost per day, with users' exposures up to 103 minutes per day. To address this issue, present mass balance-based models are already able to provide exposure estimates for both users and populations. This screening study shows large variations of up to 10 orders of magnitude in impact across both chemicals and product combinations, demonstrating that prioritization based on hazard only is not acceptable, since it would neglect orders of magnitude variations in both product usage and exposure that need to be quantified. To address this, the USEtox suite of mass balance-based models is already able to provide exposure estimates for thousands of product-chemical combinations for both users and populations. The present study calls for more scrutiny of most impacting chemical-product combinations, fully ensuring from a regulatory perspective consumer product safety for high-end users and using protective measures for users.

Dataset Information

Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals.

Publications

Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals.

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