Targeted functional genomics represents a powerful approach for studying gene function in vivo and in vitro. However, its application to gene expression studies in human mast cells has been hampered by low yields of human mast cell cultures and their poor transfection efficiency. We developed an imaging system in which mast cell degranulation can be visualized in single cells subjected to shRNA knockdown or CRISPR–Cas9 gene editing. By using high-resolution confocal microscopy and a fluorochrome-labeled avidin probe, one can directly assess the alteration of functional responses, i.e., degranulation, in single human mast cells (10–12 weeks old). The elimination of a drug or marker selection step avoids the use of potentially toxic treatment procedures, and the brief hands-on time of the functional analysis step enables high-throughput screening of shRNA or CRISPR–Cas9 constructs to identify genes that regulate human mast cell degranulation. The ability to analyze single cells substantially reduces the total number of cells required and enables the parallel visualization of the degranulation profiles of both edited and non-edited mast cells, offering a consistent internal control not found in other protocols. Moreover, our protocol offers a flexible choice between RNA interference (RNAi) and CRISPR–Cas9 genome editing for perturbation of gene expression using our human mast cell single-cell imaging system. Perturbation of gene expression, acquisition of microscopy data and image analysis can be completed within 5 d, requiring only standard laboratory equipment and expertise.
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The datasets generated during and/or analyzed during the current study are available from the corresponding author(s) upon reasonable request; an example dataset is provided as Supplementary Data.
The custom Fiji macros described in the main text of this study can be accessed and used by readers without restriction. The code in this manuscript has been peer-reviewed.
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We thank C. Liu for technical support and A. van Stigt for helpful discussions regarding MRGPRX2 expression and gene knockout in human mast cell models. We thank P. Dubreuil (Inserm, Marseille, France) for the kind donation of CHO cells. This research was supported by NIH grants U19AI104209, R01 AR067145 and R01 AI32494 (to S.J.G.) and a Marie Skłodowska-Curie Individual Fellowship (H2020-MSCA-IF-2016 no. 749629), the European Research Council (ERC-2018-STG no. 802041), the INSERM ATIP-Avenir program (N.G.) and the Lung Foundation Netherlands (grant 22.214.171.124 to R.W.H). J.F. was supported by a Fulbright Fellowship (financed by the Netherland-America Foundation). R.S. was supported by an NWO Veni Fellowship (grant 91617114) and an Erasmus MC Fellowship.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Hydar Ali and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Gaudenzio, N. et al. J. Clin. Invest. 126, 3981–3998 (2016): https://doi.org/10.1172/JCI85538
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Folkerts, J., Gaudenzio, N., Maurer, M. et al. Rapid identification of human mast cell degranulation regulators using functional genomics coupled to high-resolution confocal microscopy. Nat Protoc (2020). https://doi.org/10.1038/s41596-019-0288-6