title: Vectors for Genetically-Encoded Tags for Electron Microscopy Contrast in Drosophila.
abstract: BACKGROUND
  One of the most notable recent advances in electron microscopy (EM) was the development of genetically-encoded EM tags, including the fluorescent flavoprotein Mini-SOG (Mini-Singlet Oxygen Generator). Mini-SOG generates good EM contrast, thus providing a viable alternative to technically-demanding methods such as immuno-electron microcopy (immuno-EM). Based on the Mini-SOG technology, in this paper, we describe the construction, validation and optimization of a series of vectors which allow expression of Mini-SOG in the Drosophila melanogaster genetic model system.
  
  
  RESULTS
  We constructed a Mini-SOG tag that has been codon-optimized for expression in Drosophila (DMS tag) using PCR-mediated gene assembly. The photo-oxidation reaction triggered by DMS was then tested using these vectors in Drosophila cell lines. DMS tag did not affect the subcellular localization of the proteins we tested. More importantly, we demonstrated the utility of the DMS tag for EM in Drosophila by showing that it can produce robust photo-oxidation reactions in the presence of blue light and the substrate DAB; the resultant electron micrographs contain electron-dense regions corresponding to the protein of interest. The vectors we generated allow protein tagging at both termini, for constitutive and inducible protein expression, as well as the generation of transgenic lines by P-element transformation.
  
  
  CONCLUSIONS
  We demonstrated the feasibility of Mini-SOG tagging in Drosophila. The constructed vectors will no doubt be a useful molecular tool for genetic tagging to facilitate high-resolution localization of proteins in Drosophila by electron microscopy.
authors.affiliation: Developmental Signalling Unit, Research Lab 1, Level C, Okinawa Institute of Science and Technology, Tancha 1919-1, Onna-son, Kunigami-gun, 904-0412 Okinawa Japan.
authors.forename: Marco Man Kin
authors.initials: MM
authors.lastname: Tsui
authors.affiliation: Developmental Signalling Unit, Research Lab 1, Level C, Okinawa Institute of Science and Technology, Tancha 1919-1, Onna-son, Kunigami-gun, 904-0412 Okinawa Japan.
authors.forename: Anri
authors.initials: A
authors.lastname: Itoh
authors.affiliation: Developmental Signalling Unit, Research Lab 1, Level C, Okinawa Institute of Science and Technology, Tancha 1919-1, Onna-son, Kunigami-gun, 904-0412 Okinawa Japan ; Faculty of Medicine, Cairo University, Cairo, Egypt.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Amgad
authors.affiliation: Developmental Signalling Unit, Research Lab 1, Level C, Okinawa Institute of Science and Technology, Tancha 1919-1, Onna-son, Kunigami-gun, 904-0412 Okinawa Japan.
authors.forename: Shao-Fang
authors.initials: SF
authors.lastname: Wang
authors.affiliation: Developmental Signalling Unit, Research Lab 1, Level C, Okinawa Institute of Science and Technology, Tancha 1919-1, Onna-son, Kunigami-gun, 904-0412 Okinawa Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Sasaki
keywords: Drosophila; Electron microscopy; Imaging; Mini-SOG
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