1.4 Comparison
of FP Variants
in Mammalian Cells
MCP-FP variants displayed distinct performance in yeast, and we
thus tested how these variants [74, 76, 78–81] performed in mammalian cells (Fig. 3 and Table 1). Transient transfections of the
different NLS-MCP-FP variant constructs were evaluated in a
HeLa H9 cell line without RNA reporter gene (Fig. 3, top panels).
A diffuse fluorescent signal was seen in the nucleus, indicating a
good solubility of all NLS-MCP-FP variant constructs. When the
various NLS-MCP-FP were expressed in a derivative of this cell line
expressing the HIV-1 MS2x64 RNA reporter (Figs. 3 and 4,
Fig. 3 Comparison of MCP-FP variants in mammalian cells. Images are maximum intensity projections from
HeLa Flp-in cells transfected with the indicated NLS-MCP-FP variant constructs. Top panels: Images are from
cells expressing the indicated NLS-MCP-FP variants without RNA reporter (parental HeLa H9 AAVS1-Tat cell
line). Middle panels: Images are from cells expressing the indicated NLS-MCP-FP variant in HeLa H9 AAVS1Tat cell line expressing the HIV-1 MS2x64 reporter. Bottom panels: Images are smFISH signals obtained with
probes against the MS2 tag. Scale bar 5 μm. Contrast adjustment is identical for all images of the top and
middle panels
Table 1
List of the FP variants
NLS-MCP-FP variants
FP plasmid from
References for the FP
eGFP
Gift from Lionnet T./Singer RH
Cormack et al. (1996) [78]
mEGFP
Addgene #54610
Zacharias et al. (2002) [79]
sfGFP
Addgene #60907
Pedelacq et al. (2006) [80]
GFPenvy
Addgene #60782
Slubowski et al. (2015) [76]
muGFP
Gift from Scott DJ
Scott et al. (2018) [74]
mNG
pUC57-mNeonGreen
Shaner et al. (2013) [81]
Imaging Single mRNAs in Living Eukaryotic Cells
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