Over Expression in Drosophila – The UAS-GAL4 Binary System

The reason for Drosophila melanogaster being a very successful model system when compared to other equally beautiful model organisms in the field will be its short life cycle, cheap to maintain in lab, availability of BALANCER CHROMOSOME’S , many offspring’s, feature-rich morphology (various genetic markers) and not to forget wide array of genetic tools.

The last feature tilts the balance slightly in favor of Drosophila over its other counterparts in the field.

One could perform loss of function studies using various ways (P-element mutagenesis, EMS, UAS RNAi and CRISPR) and generating somatic clones and germ line clones are not that difficult with the available tools (using flp-frt and OVOD mutations). Making a transgenic fly is straight forward and well established (an experienced worker can inject more then five constructs in a day).

Another boon to the system of Drosophila is the tool for over expression (Gain of function studies) for targeted gene expression. Using UAS GAL4 system one can over express a gene of interest in tissue of choice. In my view UAS GAL4 system, Gene transformation along with Balancer chromosomes (these chromosomes have many inverted repeats in order to prevent recombination among homologous chromosomes) played a very important role is making Drosophila a very successful model organism. All this is possible due to great minds and lot of hard work of various researchers.

Background:

Gal4 is a protein of 881 amino acid long identified in the yeast Saccharomyces cerevisiae as a regulator of genes which is induced by galactose.The Gal4 protein binds to a sequence called Upstream Activating Sequences (UAS) element, analogous to an enhancer element defined in multicellular eukaryotes.In 1988 Fischer et al demonstrated that Gal4 protein is able to induce reporter gene under the control of UAS sequence even in Drosophila and Gal4 didn’t have any deleterious effects in fruit fly.These two things laid the foundation for the remarkable discovery in the field by Andrea Brand and Norbert Perrimon in 1993.

The article which published in Development in 1993 describes the UAS GAL4 system as a tool for targeted gene expression in fruitfly Drosophila.Targeting gene expression in a temporal and spatial fashion has proven to be one of the most powerful techniques for addressing gene function in vivo.

This system works this way:

• The responder (Gene ‘X’ to be activated) is under the control of UAS sequence. Here five UAS sites are tandemly arranged and these sequenes needs GAL4 to be activated, hence they will remain transcriptionally silent.
• Flies which can over express toxic products can be maintained in this way. One need to clone the gene of interest whose function need to studied in pUAST , transformation vector.
• This is a modified P-element with five UAS sites and Multiple cloning sites . Once you clone the gene of interest in this vector, it can be injected in the embryo for generating a Transgenic fly which can express the gene of interest.
• In order to activate transcription , the flies carrying the UAS-GENE ‘X’ need to be crossed to flies with GAL4 gene which would be under the control of a enhancer.
• The GAL4 protein is expressed depending on the expression pattern of enhancer cloned.
• Then the resulting progeny from the UAS – GAL4 cross, will over express or mis-express the gene ‘X’ in a GAL4 dependent manner.
• An additional strength of the system arises from the ability to target expression of any responder in a variety of spatial and temporal fashions by mating it with distinct GAL4 drivers which are available in plenty in Drosophila community.
• If you don’t find the GAL4 driver of interest you have vectors (pGaTB), where promoters of choice can be subcloned upstream of GAL4 at the unique restriction site, which can be then used for making transgenic fly for directing gal4 expression in cells of interest .
• From the days of Brand and Perrimon discovery various modification took place in the vector for transformation, the recent being you can direct the transgene to one particular locus in genome using attp -attB system from Viruses.

Reference:

Brand, A.H., Perrimon, N. (1993).
Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118(2): 401–415.

For map and sequences of pUAST ,PGawB, pPTGAL click here