Limitations of Green Fluorescent Protein as a Cell Lineage Marker

Abstract

The enhanced green fluorescent protein (GFP) reporter has been widely adopted for tracking cell lineage. Here, we compare three transgenic mouse strains in which GFP is considered "ubiquitously expressed," with the GFP transgene under control of the chicken ␤-actin (CBA) or human ubiquitin C (UBC) promoter. We compared the expression of GFP using flow cytometry, direct tissue fluorescence, and immunostaining with multiple commercially available anti-GFP antibodies. Mice of CBA-GFP strain 1Osb have strong but variegated expression of GFP in adult liver, kidney, small intestine, and blood. Mice of CBA-GFP strain Y01 have the highest proportion of GFP-positive peripheral blood cells yet limited GFP expression in liver, intestine, and kidney. UBC-GFP mice express GFP only weakly in solid organs and variably in blood. Direct fluorescent detection of GFP in formalin-fixed, paraffin-embedded tissue sections was the simplest approach, but it was useful only in high-expressing strains and potentially subject to artifact because of tissue autofluorescence. Immunofluorescence using either primary goat or primary rabbit antibodies was much more sensitive and allowed better discrimination of authentic signal from autofluorescence. Immunohistochemical staining was less sensitive than direct fluorescence or immunofluorescence and was subject to false-positive signal in the small intestine. In conclusion, there is considerable variability of expression within and between GFP transgenic strains. None of the tested strains gave truly ubiquitous GFP expression. A detailed analysis of GFP expression in one's tissues of interest must guide the choice of reporter mouse strain when GFP is used as a marker of cell lineage or donor origin.

A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification

“…The implanted esophagi were harvested at 12, 24, and 48 hours after implantation, fixed by 4% PFA, and processed for histology and immunostaining or snap-frozen and processed for fluorescent imaging. GFP expression was detected in paraffin-embedded samples using immunofluorescence with an anti-GFP antibody (Invitrogen) (40).…”

Cutting Edge: Broad Expression of the FoxP3 Locus in Epithelial Cells: A Caution against Early Interpretation of Fatal Inflammatory Diseases following In Vivo Depletion of FoxP3-Expressing Cells

“…We also observed no specific signal in kidney, liver, intestine, and heart in the FoxP3 EGFP mice (data not shown). It is well established that immunofluorescence with anti-GFP Abs, a strategy used here, is more sensitive and better able to discriminate between authentic GFP expression and autofluorescence (12). Relying on direct GFP visualization may explain the failure of others to observe FoxP3 locus activation in the thymic epithelial cells (13).…”

Isolation of Mesenchymal Stem Cells (MSCs) from Green Fluorescent Protein Positive (GFP+) Transgenic Rodents: The Grass Is Not Always Green(er)

“…There is considerable variability in GFP expression among different transgenic animal strains, and even inconsistency in GFP expression among various tissues within a given animal [4]. Additionally, the optimal method of GFP detection is dependent on the tissue or cell type containing the gene, along with the method used to detect the presence of GFP (microscopy, fl ow cytometry, etc.…”