Nuclear tagging in D. melanogaster. (A) Schematic representation of the D. melanogaster nuclear targeting fusion (NTF) transgene, consisting of the mesoderm-specific twi promoter, the RanGap gene tagged with 3xFLAG, biotin ligase recognition peptide (BLRP), and mCherry. A second twi promoter drives transcription of E. coli biotin ligase gene (birA). (B,C) Confocal imaging of mCherry localization in a living stage 13 embryo. Anterior is to the left. (B) Mesodermal expression pattern is evident in somites. Some autofluorescence was also observed in particles in the yolk sac at this stage. (C) mCherry is present in the nuclear envelope and in the surrounding cytoplasm. (D–G) Detection of biotin and Flag epitopes in a fixed stage 7 embryo. (D) DAPI. (E) Anti-Flag. (F) Streptavidin, detecting prominent mesodermal signal from NTF and weak peripheral signal from endogenous biotinylated proteins. (G) Merge of D (blue), E (yellow), and F (green). (H–J) Localization of NTF to larval salivary gland nuclei in Actin5C-GAL4; UAS-NTF flies. (H) Actin 5C/+ cells, not expressing NTF. (I) Actin 5C/+; UAS-NTF/+ cells. (J) Actin 5C/+; UAS-NTF/+ nucleus, broken free of cytoplasm. mCherry fluorescence is shown in red, DAPI staining in blue. (K–N) Affinity-purified nuclei (arrowheads) bound to an anti-Flag-coated bead. (K) DAPI. (L) mCherry. (M) Streptavidin. (N) Merge of K (blue), L (red), and M (green). Note that anti-Flag-coated beads are 25× larger than streptavidin-coated beads.
