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.
Highlights
Interleukin-7 (IL-7) is a cytokine essential for T cell development and peripheral T cell homeostasis, and is mainly expressed in the epithelial and stromal cells [1,2,3,4,5]
The IL-7 50 untranslated region (UTR) contains several upstream ATGs, which are known to inhibit the translation of the authentic ATG start codon in IL-7 and many other mRNAs [28,29,30,31]
EGFP protein production from enhanced green fluorescent protein (eGFP) mRNA containing upstream ATGs (uATGs) would be very weak, even though the eGFP mRNA exists. To avoid this potential problem, we intentionally designed the left homology arm (LHA) to remove several uATGs by not including part of the 50 UTR and mutating the uATGs, without affecting IL-7 promoter sequences such as the interferon stimulated response element (ISRE) (S1 Fig) which can respond to IFN-γ, and designed the right homology arm (RHA) to start from the authentic ATG codon [9, 32]
Summary
Interleukin-7 (IL-7) is a cytokine essential for T cell development and peripheral T cell homeostasis, and is mainly expressed in the epithelial and stromal cells [1,2,3,4,5]. The regulatory mechanisms mediating the expression of IL-7 have not been well studied. IL-7 Reporter Cells Useful for High-Throughput Chemical Screening. Mechanism for IL-7 expression is IFN-γ-dependent, which is mediated by the interferon stimulated response element (ISRE) in the IL-7 promoter [7, 9]. IL7 has great therapeutic potential in treating diverse immunodeficiency-related diseases [12, 13]. The cost of producing IL-7 protein for clinical application is much higher (>10-fold) than that of producing standard chemical drugs [14]. Identification of effective IL-7-inducing chemicals would be of great value in medicine
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
