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bioRxiv · 10.1101/2023.09.06.556381

PUFFFIN: A novel, ultra-bright, customisable, singleplasmid system for labelling cell neighbourhoods

Lebek, T.·Malaguti, M.·Elfick, A.·Lowell, S.
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Abstract

Cell communication orchestrates development, homeostasis, and disease. Understanding these processes depends on effective methods to study how cells influence their neighbours. Here, we present Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a novel cell neighbour-labelling system based upon secretion and uptake of a positively supercharged fluorescent protein s36GFP. To achieve sensitive labelling, we amplified the fluorescent signal by fusing s36GFP to the ultra-bright protein mNeonGreen. Secretor cells transfer s36GFP-mNeonGreen to neighbours while retaining a nuclear mCherry, making it straightforward to unambiguously identify, isolate, and profile neighbours of secretor cells. Transfer of the s36GFP-mNeonGreen fusion occurs within minutes, enabling even relatively transient interactions to be captured or tracked over time with live-imaging or flow cytometry. To enhance flexibility of use, we incorporated HaloTag technology to facilitate colour-of-choice labelling. To further increase the range of applications, we engineered a customisable single-plasmid construct composed of interchangeable components with option to incorporate any additional transgene. This allows users to manipulate the properties of a cell, while at the same time applying a fluorescent label to the surrounding cells. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour in mammalian cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/556381v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1ea5bcaorg.highwire.dtl.DTLVardef@1303810org.highwire.dtl.DTLVardef@b0d863org.highwire.dtl.DTLVardef@4e7b1f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Lebek, T., Malaguti, M., Elfick, A., Lowell, S.. 2023-09-07. PUFFFIN: A novel, ultra-bright, customisable, singleplasmid system for labelling cell neighbourhoods. https://doi.org/10.1101/2023.09.06.556381

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