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General Information
Name
TI_set_Mi{PT-GFSTF.0,1,2}.GDP
Species
D. melanogaster
Reagent type
FlyBase ID
FBlc0000719
Project
Created by
Vector
    Title
    A set of insertions using Dhyd\Minos-based protein trap constructs Mi{PT-GFSTF.0}, Mi{PT-GFSTF.1} or Mi{PT-GFSTF.2}.
    Accessions
      Overview
      Description

      A set of transgenic insertion stocks containing a protein trap cassette, derived from Mi{MIC} insertions in coding introns using Recombinase-Mediated Cassette Exchange (RMCE); see http://flypush.imgen.bcm.tmc.edu/pscreen/technique.html. The protein trap cassette contains a EGFP-FlAsH-StrepII-TEV-3xFlag ("GFSTF") tag that is flanked on either side with a 4X(GlyGlySer) flexible linker; the mini-yellow marker is no longer present. Each insertion line contains one of three versions of the cassette (Mi{PT-GFSTF.0}, Mi{PT-GFSTF.1} or Mi{PT-GFSTF.2}), depending on the reading frame required to generate an in-frame fusion protein with the gene in which the insertion is located. This system also allows UAS/GAL4-based knockdown strategies targeting GFP-sequence containing mRNA (RNAi against GFP) or GFP fusion protein (deGradFP).

      Biosample Source
      Overview
      Strain
      Stage
      Sex
      Tissue isolated
      Other tissues studied
      Cell component
      Cell line
      Key genes
      Methods
      Sample preparation
      Reagent Details
      Methods
      Transgenic Construct used
      Protocol

      Progenitor Mi{MIC} insertions are members of the dataset TI_set_Mi{MIC}.MI.P lasmid DNA containing the donor tag cassette was injected into embryos of ~700 MiMIC strains (intronic insertions) expressing phiC31 integrase, phiC31\int. G1 progeny in which the RMCE event had occurred were identified by the loss of the y+mDint2 marker of the Mi{MIC} gene trap cassette.

      An in vivo genetic tagging method has been developed so that RMCE can be carried out without the need for embryo microinjection. Transgenic fly lines were generated which contain: 1. a construct containing the GFSTF tag cassette flanked by FRT sites (one of P{FRT(PT-GFSTF.0)}, P{FRT(PT-GFSTF.1)} or P{FRT(PT-GFSTF.2)}), 2: a heat shock-inducible source of FLPase (P{hsFLP}) and 3: a germ-line expressed phiC31 integrase (M{vas-int.B}). These flies were crossed to the appropriate Mi{MIC} insertion line, and the resulting F1 embryos were heat shocked to release the GFSTF tag cassette so that it can undergo phiC31-mediated RMCE. F2 progeny positive for the RMCE event were identified by the loss of the y+mDint2 marker of the Mi{MIC} gene trap cassette.

      Mode of Assay

      A PCR assay was used to identify RMCE events that inserted the cassette in the correct orientation to result in a fusion protein containing the internal tag.

      Data analysis

      Direct confocal imaging in live tissue is possible for most lines: expression of EGFP can be imaged in unfixed tissues in more than 90% of the samples tested.

      Comments
      Associated Data
      Size
      Associated features
      611 Insertion(s)
      Files
      Additional Information
      Synonyms and Secondary IDs (2)
      Reported As
      Symbol Synonym
      TI_set_Mi{PT-GFSTF.0,1,2}.GDP
      Name Synonyms
      A set of insertions using Dhyd\Minos-based protein trap constructs Mi{PT-GFSTF.0}, Mi{PT-GFSTF.1} or Mi{PT-GFSTF.2}.
      Secondary FlyBase IDs
        References (4)