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WIREs Dev Biol
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Using mutants, knockdowns, and transgenesis to investigate gene function in Drosophila

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Abstract The sophisticated genetic techniques available in Drosophila are largely responsible for its success as a model organism. One of the most important of these is the ability to disrupt gene function in vivo and observe the resulting phenotypes. This review considers the ever‐increasing repertoire of approaches for perturbing the functions of specific genes in flies, ranging from classical and transposon‐mediated mutageneses to newer techniques, such as homologous recombination and RNA interference. Since most genes are used over and over again in different contexts during development, many important advances have depended on being able to interfere with gene function at specific times or places in the developing animal, and a variety of approaches are now available to do this. Most of these techniques rely on being able to create genetically modified strains of Drosophila and the different methods for generating lines carrying single copy transgenic constructs will be described, along with the advantages and disadvantages of each approach. WIREs Dev Biol 2013, 2:587–613. doi: 10.1002/wdev.101 This article is categorized under: Technologies > Perturbing Genes and Generating Modified Animals Technologies > Generating Chimeras and Lineage Analysis Technologies > Analysis of Cell, Tissue, and Animal Phenotypes

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The Flp/FRT system for generating homozygous mutant clones marked by the loss of GFP. Homozygous mutant clones are induced by expressing the Flp‐recombinase in the tissue of interest to catalyze site‐specific recombination between FRT sites at identical positions near the centromere on homologous chromosomes. When this occurs in a cell in the G2 phase of the cell cycle and the resulting chromatids segregate appropriately at the next mitosis, one of the daughter cells will inherit two copies of the mutant chromosome arm and no copies of GFP to give rise to a clone of mutant cells that are marked by the absence of GFP expression.

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Alternative bipartite transcriptional activation systems for controlling gene expression. (a) In the LexA system, the DNA‐binding domain of the Escherichia coli LexA protein is fused to the VP16 transcriptional activation domain. This fusion protein activates transgenes downstream of multiple copies of the lexA‐binding site and a basal promoter (Lexaop). (b) The Tet‐Off system consists of the E. coli Tetracyclin repressor fused to the Herpes simplex virus VP16 activation domain to create a Tetracycline‐controlled transactivator protein (tTA) that binds to TetO sites to activate transcription. Addition of Tetracycline blocks transcription by preventing the binding of tTA to TetO. (c) The Tet‐On system uses a modified version of tTA (rtTA) that has been mutated so that it binds to TetO in the presence rather than the absence of antibiotics. This means that the addition of an antibiotic, such as doxycycline, turns the target genes on rather than off. (d) The Q system is based on the Neurospora crassa pathway that controls quinic acid metabolism. The qa‐1F factor (QF) activates genes downstream of a Q upstream activating sequence (QUAS). QF‐dependent activation is blocked by the binding of the qa‐1s protein (QS) to the QF transactivation domain, but this repression is relieved in the presence of quinic acid.

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The Gal4 system and its derivatives. (a) A P{Gal4} insertion expressing Gal4 from the P‐element promoter under the control of a nearby genomic enhancer. Gal4 protein binds to the Upstream Activation Sequence of a UAS‐transgene to activate its transcription in cells in which it is expressed. (b) In the split Gal4 system, different enhancers drive the independent expression of leucine zippers fused to the Gal4 DNA‐binding domain (Zip‐Gal4DBD) and the p65 transcriptional activation domain (p65AD‐Zip). In cells that express both constructs, the leucine zippers drive the dimerization of the two domains to produce a composite transcription factor that activates UAS‐transgene expression. This system gives more restricted patterns of UAS‐transgene expression than normal Gal4, as the transgenes are only activated in the overlap between the Zip‐Gal4DBD and p65‐Zip expression patterns. (c) Gal80ts binds to the transcriptional activation domain of Gal4 at the permissive temperature of 18°C and represses the transcription of UAS‐transgenes. Upon shifting flies to the restrictive temperature of 29°C, Gal80ts is inactivated to allow Gal4‐dependent UAS‐transgene expression. (d) In the Geneswitch system, the hormone‐binding domain of the Progesterone receptor (PR) is fused to Gal4. In the absence of hormone, the Gal4‐PR fusion protein is kept in the cytoplasm through the binding of Hsp70 to the PR domain. Treating animals with a hormone analog that binds to the PR domain, such as RU486, releases the Gal4‐PR fusion protein from the cytoplasmic tether, allowing it to enter the nucleus and activate transcription.

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Generating site‐specific mutations with TALEN nucleases. (a) The TALEN nuclease is constructed by selecting the appropriate sequence of TALE domains, each of which binds to a single base pair of DNA. The series of tandem TALE domains is then cloned into a vector that contains the N and C‐terminal TALE domains, a nuclear localization signal and a Fok1 nuclease domain. Targeting a specific site in the genome requires two TALENs that recognize sequences on opposite strands of the DNA about 16 base pairs apart. The binding of both TALENs allows the Fok1 nuclease domains to dimerize and cut the genomic DNA. Repair of this double‐stranded DNA break by the error‐prone, non‐homologous end‐joining pathway introduces mutations into the targeted gene. (b) Examples of the sorts of mutations produced by TALEN nuclease treatments. (c) mRNAs encoding a pair of TALENS are injected into the pole plasm to target the nuclease to the germline. The progeny of injected animals are then screened by PCR for new TALEN‐induced mutations.

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Gene targeting by ‘ends in’ homologous recombination. Ends in gene targeting differs from ‘ends out’ in that the single I‐SceI site in the construct lies within the region that is homologous to the endogenous locus. This results in the production of a linear DNA fragment that recombines with the endogenous gene with its ends in. This produces a tandem duplication of the gene, in which the two copies are separated by the marker gene from the targeting construct. The duplication is then resolved by crossing in a source of I‐CreI, which cuts between the marker gene and one of the tandem copies of the locus to produce a double‐stranded DNA break that is repaired by homologous recombination between the two tandem copies of the gene. If a mutation is introduced into the targeting construct, it will be present in one of the two tandem copies of the gene produced by the initial homologous recombination event. A proportion of the recombinants produced by resolving the duplication with I‐CreI will carry this mutation.

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A positive/negative selection scheme for ‘ends out’ gene targeting. This approach is identical to standard ‘ends out’ gene targeting, except that UAS‐Reaper is included in the targeting vector between the two I‐SceI sites, but outside the two homology regions. UAS‐Reaper is lost during homologous recombination into the correct site because it lies outside of the two homology regions that recombine with the endogenous locus. It is inserted into the genome during most non‐homologous integration events, however, and is present in the original targeting vector. When a source of the Gal4 transcriptional activator is crossed in to the progeny, it will bind to the UAS to activate the expression of Reaper, which causes rapid death, thereby eliminating flies with non‐homologous integrations.

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Gene targeting by ‘ends out’ homologous recombination. The targeting vector is inserted into the genome by standard transformation techniques. Heatshock‐inducible Flp‐recombinase and I‐SceI constructs are crossed into flies carrying this targeting construct. Upon heatshock treatment, Flp catalyzes recombination between the FRT sites to excise the intervening region as a circular plasmid, which is then linearized by I‐SceI cleavage at two sites. The homologous regions of this linear DNA fragment then recombine with the endogenous gene to disrupt it by insertion of the marker gene (mini‐white).

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Germ‐line transformation by recombination‐mediated cassette exchange (RMCE). DNA constructs can be very efficiently inserted into a defined site in the genome using phiC31 to catalyze RMCE. The targeting construct (red) is cloned between two attB sites with opposite orientations, and is injected into the pole plasm of flies expressing phiC31 recombinase that carry a genomic target site containing two inverted attP sites. The most common target sites are insertions of the MiMIC element, which carries yellow+ as a visible marker. PhiC31 then catalyzes recombination between the both sets of attB and attP sites to insert the targeting construct in either orientation in place of the eGFP and yellow+ in the MiMIC element. One important advantage of this approach is that one does not need to include a selectable marker in the targeting construct, as the loss of yellow+ can be used to identify the products of RMCE. RMCE can also be performed using pairs of incompatible lox and FRT sites.

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Methods for temporally and spatially controlled knockdown of protein function. (a) The deGradFP technique. The Gal4/UAS system is used to drive the expression of a single chain antibody (a nanobody) directed against GFP fused to the F‐box domain of Slimb. The nanobody binds to the GFP‐tagged protein, while the Slimb F‐box recruits the rest of the SCF ubiquitin ligase complex. This leads to the poly‐ubiquitinylation of the GFP‐tagged protein, which is targeted for degradation by the proteasome. (b) Chromophore Assisted Laser Inactivation (CALI, also called FALI for fluorophore‐assisted laser inactivation): Intense laser illumination of GFP chromophore leads to the production of reactive oxygen species that inactivate any proteins within 3–5 nm, which is mainly the protein to which the GFP is fused. (c) An example of CALI on a Myosin Regulatory Light Chain‐GFP fusion (from Monier et al.). A focused laser beam was used to inactivate MRLC‐GFP on one side of the contractile ring of a dividing cell. This blocks the activity of myosin on this side of the contractile ring and arrests the progression of the cleavage furrow locally. CALI on a control protein that localizes to the contractile ring, but is not required for cytokinesis, had no effect.

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The MARCM system for generating positively marked mutant clones. Mosaic analysis with a repressible cell marker (MARCM) is a technique for generating homozygous mutant clones that are positively marked by the presence of GFP. Homozygous mutant clones are generated using the Flp/FRT system (see Figure ), with a constitutively expressed Gal80 construct on the non‐mutant chromosome arm. In cells that have not undergone recombination, Gal80 represses Gal4 to prevent the expression of UAS‐GFP. After recombination and cell division, the homozygous mutant cells lack the Gal80 transgene so Gal4 can turn on GFP expression. Note that the Gal4 and UAS‐GFP transgenes do not need to be on the chromosome arm that carries the FRT site and the mutation of interest, but can be on one of the other choromosomes. Gal80 protein can perdure for some time after the mutant clones are generated, so the youngest clones may not yet be marked by GFP expression.

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