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First published online September 28, 2007
doi: 10.1242/10.1242/dev.005686

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Transgenesis upgrades for Drosophila melanogaster

¹ Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA.
² Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
³ Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
⁴ Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Drosophila transgenesis. white+ transgene DNA (red) is injected into generation zero Drosophila embryos (G0) of less than 1 hour old, which have been obtained from a parental (P) generation. The early developmental stages of Drosophila embryos are characterized by rapid nuclear divisions that occur without accompanying cell divisions, creating a syncytium. Prior to cellularization, pole cells (black) bud off at the posterior end. For germ line transmission to occur, the transgenic DNA must be taken up into the pole cells that are fated to become germ cells. Transgenic DNA integrated into a pole cell (red pole cell) can be transmitted from one generation (G0) to the next (G1 progeny). The resulting integration events are identified using an appropriate marker, such as as white+. When used in a mutant white- strain, this transgene marks transgenic flies by giving them a darker eye color (see Table 2 and Box 3 for more information on the markers used in fly transgenesis).

View larger version (10K): [in this window] [in a new window]   Fig. 2. Binary vector/helper transposon transformation system. (A) Active transposons are mobile elements that consist of two inverted terminal repeats (black) that flank an open reading frame encoding a transposase. Both features are required for transposition. The inverted repeats are commonly called 5' or Left (L) and 3' or Right (R). Transposition results in a duplication of the insertion site (blue). (B) Transposon and transposase can be separated, resulting in a binary vector/helper transposon transformation system that allows the regulated transposition of transgenes into the genome. Transposition events are identified by dominant markers (green, and see Table 2 and Box 3).

View larger version (11K): [in this window] [in a new window]   Fig. 3. Transgene coplacement. Two inserts (1 and 2, red), each containing a cloned fragment, such as a genomic rescue fragment, are integrated into a P element that contains appropriately positioned loxP (yellow) and FRT (pink) sites. Cre recombination results in the removal of insert 2, whereas FLP recombination results in the removal of insert 1, positioning either insert in the same orientation at the same locus (indicated in blue), thereby neutralizing position effects. In each case, recombination events are identified by the loss of a dominant marker (green).

View larger version (38K): [in this window] [in a new window]   Fig. 4. FLP remobilization. (A) FLP remobilization technique. A donor transposon contains a transgenic insert (red) together with a marker (1) flanked by two FRT sites. An acceptor transposon, at a desired locus, contains a second marker (2) and one FRT site. Remobilization of the donor transposon by FLP results in the excision of its transgene and its potential integration into the FRT site of the acceptor transposon. This remobilization can be followed through changes in expression of marker 1, such as white, that occur because of changes in position effects (from yellow in the original site to orange in the acceptor site). Different donor transposons, each containing different transgenes, can be targeted to the same acceptor, thereby neutralizing position effects. (B) Split white+ marker strategy. The white+ marker is divided into two parts: 5'-white+ (5') and 3'-white+ (3'). Neither part can produce eye pigmentation alone (indicated in gray). Recombination between appropriately localized recombination sites, FRT in this case, results in white+ reconstitution and its expression (orange). (C) Integration of the split white+ marker strategy into the FLP remobilization technique. The correct remobilization and integration of the transgene (red) are identified by white+ reconstitution (orange). Marker 2 (yellow) identifies donor transgenes. (D,E) DrosDel elements P{RS5} and P{RS3}. FLP-mediated recombination at (D) P{RS5} and (E) P{RS3} results in chromosomal remnants, P{RS5r} and P{RS3r}, respectively. Each contains one part of the white+ marker. Both remnants can be reconstituted through FLP remobilization of an appropriately designed donor transposon (see C).

View larger version (14K): [in this window] [in a new window]   Fig. 5. P element replacement. (A) Two P elements, an acceptor element, containing marker 1 (orange), and a donor element, containing transgenic insert (red) and marker 2 (yellow), are brought together. (B) In the presence of P transposase, the acceptor element might excise. (C) This excision might promote double-stranded gap repair through homologous recombination between the 10-20 bp footprints (pink) of the 31 bp inverted terminal repeats at the acceptor site (blue) and the similar sequence at the donor site (green). (D) This results in the integration of the donor element into the acceptor locus.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Gene targeting in Drosophila. (A) Ends-in insertional gene targeting. The donor construct, within a P element, contains a region of homology (the targeting construct, red) interrupted by a restriction recognition site for the meganuclease I-SceI and flanked by FRT recognition sites for FLP recombinase. It also contains a marker (white+) and an appropriately located restriction recognition site for the meganuclease I-CreI for a second round of homologous recombination. After P element transgenesis, a linearized episome is generated in vivo by FLP and I-SceI. Correct targeting results in white+ expression and a tandem duplication of the locus. This duplication can be reduced to single copy using I-CreI, resulting in loss of white+. (B) Ends-out replacement gene targeting. The donor construct, within a P element, contains a region of homology interrupted by a white+ marker and is flanked by restriction recognition sites for the meganuclease I-SceI and FRT recognition sites for FLP recombinase. After P element transgenesis, identified by white+, linearized targeting DNA is generated in vivo by FLP and I-SceI. Correct targeting results in a white+ phenotype and replacement of part of the locus.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Site-specific integration in Drosophila. (A) C31 integrase-mediated transgenesis using single attP docking sites. Docking sites are transposons, such as P elements (Groth et al., 2004), piggyBac (Venken et al., 2006) or Mariner (Bischof et al., 2007), that contain a single attP recombination site and a marker 1, and that are integrated into the genome. A plasmid containing an insert, marker 2 and an attB recombination site, can then integrate into the docking site when C31 integrase is provided. Correct recombination events between attP and attB are identified using marker 2. They result in two hybrid sites, attL and attR, that are no longer a substrate for C31 integrase - the reaction is therefore irreversible. (B) Cre- and FLP-mediated RMCE. Docking site transposons (with 5' and 3' transposon termini), such as P (Oberstein et al., 2005) or piggyBac (Horn and Handler, 2005) elements, contain marker 1 flanked by heterotypic direct-oriented recombination sites (RS) `RS1' (loxP or FRT, gray) and `RS2' (such as lox2272 or F3, purple). The RMCE plasmid, containing marker 2 flanked by a similar configuration of heterotypic recombination sites, can integrate when Cre or FLP is provided. Correct recombination events are identified by the absence of marker 1 and presence of marker 2. Recombination can be partial (single integration events are not shown) and is reversible. (C) C31 integrase-mediated RMCE. A docking site P element transposon (5'P and 3'P element termini) (Bateman et al., 2006) contains a marker 1 flanked by inverted attP recombination sites. The RMCE plasmid, containing insert flanked by inverted attB recombination sites, can integrate when C31 integrase is provided. Correct recombination events, between both attP and attB sites, are identified through absence of marker 1 and result in hybrid sites, attL and attR, that are no longer substrates for C31 integrase. The integrated DNA can be in either orientation (arrows).

View larger version (33K): [in this window] [in a new window]   Fig. 8. Gap repair. (A) Recombineering-mediated gap repair. Two homology arms, located at the 5' (Left, L) and 3' (Right, R) end of a genomic region of interest present in a BAC, are cloned into the desired plasmid. Restriction enzyme-mediated linearization between both homology arms and subsequent transformation in bacteria competent for recombineering functions allow the selective retrieval of the desired fragment from the BAC into the plasmid through gap repair. The resulting plasmid can be used for P transposase- (5'P and 3'P element termini) or C31 integrase-mediated transgenesis (attB site). (B) In vivo gap repair. Two homology arms, located at the 5' and 3' ends of a genomic region of interest, are cloned within a P element. After P transposase-mediated germ line transmission, the transgene is linearized in vivo between both homology arms using the meganuclease I-SceI, potentially resulting in the selective capture of the desired fragment from a wild-type chromosome through homologous recombination-mediated gap repair.

View larger version (33K): [in this window] [in a new window]   Fig. 9. Recombineering-mediated mutagenesis. (A) `Blind' mutagenesis. To perform a site-specific change in a fragment within a target plasmid, a PCR fragment or oligonucleotide that contains the desired mutation is transformed into bacteria that contain recombineering functions and a target plasmid. Recombination results in the incorporation of the desired mutation: substitution, deletion or insertion. The bacteria are then screened by PCR for the proper mutagenic event. (B) Mutagenesis using a positive/negative selectable marker. First, in the positive-selection step, a PCR fragment containing a positive (+)/negative (-) selectable marker is transformed into bacteria that contain recombineering functions and the target plasmid. Individual colonies containing the correct recombinant plasmid are then selected. Second, during the counterselection step, a PCR fragment containing the desired change, such as a tag, might replace the positive/negative selectable marker. Counterselection or negative selection may result in the selection of a correct recombinant plasmid that can be identified through PCR. (C) RecA-assisted modification. A specialized plasmid that contains a selectable marker (+), a counterselectable marker (-), RecA and a mutation flanked by two homology boxes (A and B) is transformed into bacteria. During a first recombination event, identified through selection, this plasmid can integrate through homology box A (shown) or B (not shown), resulting in a co-integrate. During a second recombination event, identified through counterselection, this co-integrate can resolve to the original plasmid (not shown) or the modified plasmid (shown).

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