Expr13397
As with the N-terminally tagged F28F8.5 [Expr13396], F28F8.5::GFP showed both nuclear and cytoplasmic localization. As expected for an extrachromosomal transgene, the expression of F28F8.5::gfp was not detected in the germline. This reporter was expressed in embryos starting at the twofold stage and continued throughout development. We did notice that F28F8.5::gfp was expressed in the excretory canal cell, a pattern not observed with the endogenously edited GFP-tagged gene.
Expr13396
The GFP::F28F8.5 pattern was ubiquitous, both nuclear and cytoplasmic from embryos to adults. Prominent nuclear localization was found in oocytes, zygotes, larvae, and adults. Cells with clear nuclear accumulation of GFP::F28F8.5 included epidermal, intestinal, pharyngeal, uterine and vulval muscle cells. The gonad expressed gfp::F28F8.5 and mitotic as well as meiotic nuclei accumulated GFP::F28F8.5 protein. Selected animals were analyzed by confocal microscopy for determination of subcellular distribution of GFP::F28F8.5. Scanning through several focal planes revealed signal in the GFP excitation/emission range in nuclei as well as in the cytoplasm of embryos, all larval stages and adults. Structures resembling gut granules were also strongly positive in the GFP recording mode. In order to distinguish between GFP-specific fluorescence and autofluorescence, we applied FLIM with an expectation that autofluorescence (such as that from gut granules) is likely to produce a signal with a short fluorescence lifetime opposed to GFP-specific fluorescence. Structures such as gut granules were clearly detected while fluorescence with a longer lifetime expected for GFP::F28F8.5 was detected in the germline, in oocytes and embryos and in most somatic nuclei of larvae as well as adult animals.