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 can negatively impact transcript detection on the Xenium Analyzer. Certain fluorophores that are sensitive to alcohol, such at GFP, 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, and if fluorescence is maintained throughout the assay workflow, this would be problematic for Xenium. For any sample expressing fluorescent reporters, we would recommend a pilot experiment.
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 is desired, and protein structure maintained, the allele may be detected with antibody staining.
Considerations for protein-based detection via IF (unsupported and low risk):
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-based reporter gene-based, RNA detection (unsupported and high risk):
There is a finite limit to the total transcripts that can be optically resolved in any particular cell. ‘Optical crowding’ is the phenomenon by which the recognition of certain genes is impaired by the presence of other genes. This is most commonly induced by the inclusion of very highly expressed genes in a gene panel. The signal of the highly expressed gene or genes exceeds the system's detection limit and drowns out genes with lower expression, leading to a decreased quality scores for genes readouts.
Reported genes, such as GFP are often very highly expressed. Extremely highly expressed genes can result in reduced overall sensitivity due to crowding. The degree to which GFP signal might obscure the detection of other genes is likely to be very sample-specific. The level of GFP (or other reporter gene) expression and also the number of cells containing the reporter gene will impact assay performance. If the reporter is low to moderate expression or only expressed in a few cells, it may be possible to design add-on probes to this reporter.
Because reporter gene expression is very sample-dependent and also the risk of including reporter genes is high, inclusion of GFP, mCherry, etc. in add-on panels is not recommended.
Considerations for custom gene inclusion can be found in the Species Standalone Custom and Advanced Custom Panel Design for Xenium In Situ Tech Note.
**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 high risk for Xenium failures.***
Products: Xenium In Situ Gene Expression