Question: Is Xenium compatible with tissues expressing a reporter protein such as GFP?
Answer: It is unknown whether Xenium is compatible with tissue sections expressing reporter proteins such as GFP, YFP, or mCherry. In general, sources of fluorescence could potentially impact transcript detection on the Xenium Analyzer for genes that have codewords that are expressed in channels similar to the reporter (assuming the reporter still retains fluorescence). Certain fluorophores that are sensitive to alcohol, such as GFP/RFP, may be lost as part of Xenium sample preparation and the endogenous fluorescence will not be retained and is unlikely to interfere with data acquisition on the Xenium Analyzer. Other fluorescent reporters may be more stable. For any sample expressing fluorescent reporters, we would recommend a pilot experiment. The concern applies to tissue autofluorescent regions, so the risk of using a sample expressing an exogenous fluorescent protein marker is on par with using a sample with high autofluorescence.
Loss of endogenous fluorescence in the Xenium Assay workflow (as described above) may not be associated with a loss of protein structure. If reporter readout is desired, and protein structure maintained, the allele may be detected with antibody staining.
Considerations for protein-based detection via IF (unsupported):
Antibody-based detection methods (immunofluorescence labeling, IF immunohistochemistry (IHC) staining) may enable reporter gene detection paired with Xenium. Please refer to the Post-Xenium In Situ Applications: Immunofluorescence, H&E, and Visium CytAssist Spatial Gene Expression Technical Note for additional details on post-Xenium Staining. We have not tested this specifically on tissue expressing reporter alleles. In summary, protein-based detection of reporter genes may be theoretically possible, but untested.
Xenium gene-based RNA detection of reporters is enabled by advanced custom probe design:
Xenium enables advanced custom probe design for exogenous marker transcripts. Researchers assume the risk of advanced custom probe performance, e.g. off-target binding and detection sensitivity. Probe design against evolutionarily distinct targets, e.g. fluorescent markers in mammalian samples, is relatively straightforward. The main concern with advanced custom probes for fluorescent markers is strong ectopic promoter driven expression of such targets and their contribution to optical crowding during RNA cycles. Panel design should tune probe set coverages appropriately for expected expression level, e.g. two to three probes for a strongly expressing marker. Detection of the fluorescent marker expression in tissue regions can then help inform interpretation of transcript detection in the tissue regions. Considerations for custom gene inclusion, Xenium v1, can be found in the Species Standalone Custom Tech Note.
The publication here shows Xenium results for murine brain tumor samples using a panel that includes advanced custom probes for eGFP and human-PDGFB transgenes. Recurrent tumor cells expressed the marker from a PDGFB-HA-SV40-GFP construct.
**While tissues expressing fluorescent proteins may be compatible with Xenium, dyes that are fluorescent are not likely to be quenched as part of the assay workflow and thus are higher risk for Xenium failures.***
Products: Xenium In Situ Gene Expression