title: 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.
authors: Lab of Molecular Immunology and Medicine (MoIM), Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors: Yeon Sook
authors: YS
authors: Cho
authors: Lab of Molecular Immunology and Medicine (MoIM), Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors: Byung Soo
authors: BS
authors: Kim
authors: Lab of Molecular Immunology and Medicine (MoIM), Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors: Chan Kyu
authors: CK
authors: Sim
authors: Department of Convergence Medicine, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul 05505, Korea.
authors: Inki
authors: I
authors: Kim
authors: Lab of Molecular Immunology and Medicine (MoIM), Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors: Myeong Sup
authors: http://orcid.org/0000-0002-8144-824X
authors: MS
authors: Lee
mesh_terms: D002460:Cell Line; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D015851:Interleukin-7 / Q000235:genetics*; D054852:Small Molecule Libraries; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D015851:Interleukin-7; D054852:Small Molecule Libraries; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins