GFPnovo2, a brighter GFP variant for in vivo labeling in C. elegans

Green fluorescent protein (GFP) is one of the most common fluorophores used to label cells and proteins in C. elegans. Previous artificial protein evolution strategy has generated a variant of eGFP, GFPnovo2, which is 3.3 times brighter than the original eGFP in the DT40 cell line (Arakawa et al, 2008). GFPnovo2 carries four mutations from the original eGFP (Y145F, V163A, S202T, L221V), and has the same excitation/emission wavelengths as the original eGFP. Here we compared the brightness of GFPnovo2, which was generated by introducing above mentioned mutations in the pSM (eGFP_unc-54 3’ utr: kind gift from Cori Bargmann) vector (Figure 1A), with that of original eGFP in the C. elegans nervous system. Under the fluorescent dissection scope, all four GFPnovo2 lines with high transmission rate (over 80%) we examined had brighter fluorescent signal at all developmental stages (late embryo to adult) than five eGFP lines with the similar high transmission rate. As axons and dendrites are thin, it is often difficult to visualize when the copy number of the transgene is low. Indeed, we observed fewer neurites when we labeled neurons with the low dose of eGFP (1ng/μl) expressed under the pan-neuronal promoter, Prab-3 (Figure 1B, top). In contrast, GFPnovo2 was considerably brighter than eGFP and nicely labeled neurites when injected at the same concentration (1ng/μl) (Figure 1B, bottom). As a result, the average signal intensities of the dorsal nerve cord, which contains only neurites, was significantly and consistently brighter in the animals expressing GFPnovo2 than those expressing eGFP (Figure 1C). The variation among the animals expressing GFPnovo2 is likely due to the mosaic nature of the extra-chromosomal array. Nevertheless, all GFPnovo2 animals had a brighter signal than eGFP animals. Similarly, we were able to label the entire axon of DA9 neuron with GFPnovo2 expressed under the DA9 specific promoter, itr-1 (Chen et al., 2018), including the axonal tip which was not easy to detect with eGFP (unpublished). We did not notice detectable photobleaching while examining animals expressing GFPnovo2 under the fluorescent compound microscope or the confocal microscope, suggesting that GFPnovo2 is at least as stable as eGFP.

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