The Gal4-UAS system: Drosophila research tool

The Gal4-UAS system is one of the most famous and useful genetic tools available in Drosophila melanogaster. This technology was first developed in yeast1,2 in the late 1980s and subsequently adapted to allow tissue-specific gene expression in flies.3 Researchers around the globe now use Gal4-UAS to turn genes on or off in almost any Drosophila tissue they desire.

How does the Gal4-UAS system work?

The operating principle of this system is simple and consists of two parts: (1) The transcriptional activator protein Gal4, which selectively binds (2) upstream activation sequences (UAS) in DNA, thereby activating transcription of a downstream gene. The system as used in flies consists of a “driver” line, expressing a Gal4-encoding transgene under the control of a tissue-specific promoter, and a Gal4-responsive “UAS” line containing a gene of interest downstream of the UAS site. A simple genetic cross of these two fly lines will yield progeny with the desired tissue-specific expression pattern of the gene of interest (see diagram below).

Gal4-UAS system Principle of operation
In this example, a female fly containing a transgene under UAS control (5x refers to 5 copies) is crossed to a male fly containing a Gal4 transgene under the control of an egg-specific promoter. The female progeny of the cross then displays egg-specific expression of the transgene, indicated by the orange signal.
Image Source: Drosophila melanogaster Oogenesis: An Overview.4

What are the advantages of the Gal4-UAS system?

The main advantage of the Gal4-UAS system is its modular design. Since each transgene (i.e. the Gal4 part and the UAS part) is carried in a separate fly line, you can maintain UAS-transgenes encoding toxic or lethal gene products without any difficulty. Only when the two components are combined is gene expression activated.

In additional, a single Gal4 driver line can be mated with thousands of different UAS lines (and vice versa) to achieve a huge variety of gene expression patterns. Genes that can be placed under UAS control are practically limitless and include fluorescently tagged proteins, double-stranded RNA (to perform RNAi), or site-specific recombinases like FLPase or Cre.

Thousands of Gal4 driver fly lines have been created, with tissue specificity ranging from ubiquitous expression to expression in just one or a few cells, depending on the choice of Gal4 promoter. Many of these lines, both Gal4 and UAS, are publicly available from Drosophila stock centers like the Bloomington Drosophila Stock Center and the Vienna Drosophila Resource Center.

Applications of the Gal4-UAS system

RNA interference (RNAi)

One of the most powerful applications of Gal4-UAS is the inducible, tissue-specific activation of RNAi for knock-down of specific mRNA transcripts. This technology is especially useful for the study of genes that are highly pleiotropic (have multiple phenotypic effects) or for which there aren’t any null mutations available.

The Drosophila Transgenic RNAi Project (TRiP) and the Vienna Drosophila RNAi Center (VDRC) are two popular consortia that have designed and store RNAi fly lines available for order. In principle, a specific mRNA transcript can be knocked down in any tissue for which there is an appropriate Gal4 line.

For example, using a fly line with Gal4 under the control of an eye-specific promoter (ey-Gal4), you could knock down the white mRNA specifically in eye tissue — producing white, pigment-free eyes as seen in the image below.

Gal4-UAS RNA interference
Using an eye-specific Gal4 driver to knock down the white mRNA in eye tissue (Control on left, white knock-down on right). White is a protein partially responsible for the red eye pigmentation of D. melanogaster.
Image source: Nature Communications.5

Expression of fluorescently-tagged proteins

Another useful application of Gal4-UAS is to express fluorescently-tagged proteins in tissues of interest. This technique can be used to visualize the locations of proteins within different tissues, or even study how they behave in live cells. The images below are captured from fly larvae expressing the protein CD8 tagged with GFP (green fluorescent protein) in different tissues according to the specific Gal4 driver used.

Gal4-UAS fluorescent protein
Expression patterns of different Gal4 drivers in the developing larval central nervous system (CNS). elav-Gal4 (top panel) drives expression of UAS-CD8-GFP in all neurons of the CNS. cha7.4-Gal4 (middle panel) drives expression of UAS-CD8-GFP in cholinergic inter- and sensory neurons. ppk-Gal4 (bottom panel) drives expression of UAS-CD8-GFP in a subset of nociceptive neurons. Image source: J Neurograd Neurosci Educ. 6

An especially ingenious application of the Gal4-UAS system is the construction of the “Brainbow,” a project to visually map the various cell lineages in the D. melanogaster brain.7 You can see the principle of its design in the top panel below: UAS sites are engineered upstream of transgenes encoding three different fluorescent proteins. When those transgenes are randomly recombined during fly brain development (via the three different loxP sites), different varieties of fluorescently colored neurons can be generated as seen in the bottom panel below.

Principle of design of the fluorescently labeled “Brainbow” using Gal4-UAS engineering.

References

  1. Kakidani, Hitoshi and Ptashne, Mark (1988). “GAL4 activates gene expression in mammalian cells”. Cell. 52 (2): 161–167. doi:10.1016/0092-8674(88)90504-1
  2. Webster, N.; Jin, J. R.; Green, S.; Hollis, M.; Chambon, P. (1988). “The yeast UASG is a transcription enhancer in human HeLa cells in the presence of the GAL4 trans-activator”. Cell. 52 (2): 169–78. doi:10.1016/0092-8674(88)90505-3
  3. Brand, A. H. and Perrimon, N. (1993). “Targeted gene expression as a means of altering cell fates and generating dominant phenotypes”. Development. 118 (2): 401–415. doi:10.1242/dev.118.2.401
  4. McLaughlin J.M., Bratu D.P. (2015) Drosophila melanogaster Oogenesis: An Overview. In: Bratu D., McNeil G. (eds) Drosophila Oogenesis. Methods in Molecular Biology, vol 1328. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2851-4_1
  5. Qiao, HH., Wang, F., Xu, RG. et al. An efficient and multiple target transgenic RNAi technique with low toxicity in Drosophila. Nat Commun 9, 4160 (2018). https://doi.org/10.1038/s41467-018-06537-y
  6. Berni J, Muldal AM, Pulver SR. Using Neurogenetics and the Warmth-Gated Ion Channel TRPA1 to Study the Neural Basis of Behavior in Drosophila. J Undergrad Neurosci Educ. 2010 Fall;9(1):A5-A14. Epub 2010 Oct 15. PMID: 23494686; PMCID: PMC3597422.
  7. Hampel, S., Chung, P., McKellar, C. et al. Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns. Nat Methods 8, 253–259 (2011). https://doi.org/10.1038/nmeth.1566

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