The wide applicability and everyday use of the system can be attributed to its simple design and availability of lots of reagents ( Huge collection of GAL4 , UAS-lines and new stocks are frequently added).
One of the important aspect to keep in mind while setting up GAL4-UAS crosses is to understand the relationship between GAL4 protein and temperature.
GAL4 protein and temperature :
The activity of Gal4 protein is dependent on temperature. In Drosophila, The minimal intensity of Gal4 protein is observed at 16°C and maximal activity is observed at 29°C without much affecting the fertility and viability due to growth at higher temperature.
This gives the researchers a range of temperatures from 16°C to 29°C to set up the experiments, thereby increasing the flexibility of the system. Just by changing the temperature, the level at which GAL4 driver and eventually the responder construct ( UAS ) should express, can be controlled.
This property of GAL4-UAS system can be exploited for studying function of a gene at post embryonic stages of development.
If a GAL4 driver used in an experiment to transcribe a responder ( in this case UAS-GENE X ) has a strong ubiquitous expression at all stages of development (actin-GAL4 or Tubulin-GAL4) , might sometimes leads to undesired lethality before the first larval stage .
This will hamper to study the function of Gene-X (if any) at post embryonic stages – Larval, Pupal and Adult. By maintaining the cross of the ubiquitous expressing GAL4 and UAS-GeneX at 16°C will allow the embryos to escape lethality.
Once the movement of larvae is observed, the cross should be shifted to regular temperature of 25°C and any possible phenotype at post embryonic stages can be examined for deciphering the function of Gene X in later stages of Drosophila development.
One Important thing to remember while performing any UAS-GAL4 experiment is to maintain a constant temperature and not to fiddle with temperatures, beginning from the day of setting the cross till the day of scoring the larvae, pupae or adults for the phenotype.
If the cross experience variable temperatures then the phenotype observed may not be a reliable and difficult to reproduce. However in case of a lethality is observed at embryonic stages (either due to strong expression of either GAL4 driver or responder), temperature shift as mentioned above should be carried out.
GAL4 and GAL80 :
The Saccharomyces cerevisiae Gal80 protein acts as repressor of GAL4 protein by directly binding to its carboxy terminus and hence prevents GAL4 mediated transcriptional activation. This interaction of GAL4 and GAL80 can be employed to refine the expression pattern of GAL4-dependent transgenes. Here we describe two ways how GAL80 protein can be used to study development at post embryonic stages of Drosophila:
GAL80 protein under the control of a ubiquitous promoter such as Tubulin (Tub-pGAL80) is used in a combination of two other classic genetic tools, UAS-GAL4 and Flp-FRT somatic recombination, to restrict transgene expression in tissue of our interest and generate cell populations of different identities ( Genetic mosaics).
Mutations in some genes results in lethality when in homozygous condition ( in two copies ) at early developmental stages, which prevents us to study its function at later stages.
By combining FRT-UAS-GFP fly line with mutation of interest and then crossing to another fly line with a flippase gene tagged downstream of heat shock promoter, GAL4 driver and FRT-TubpGAL80.This set up will allow us to generate genetic mosaic of cells and study the fate of cells in complete absence of the gene of interest in clones, which is otherwise not possible due to lethality at embryonic stages.
Mitotic recombination is induced by giving heat shock at 37°C for around 1hr to induce flippase. The fliappase produced binds to Flippase recognizing targets for initiation of recombination events to a give range of genetic mosaics for the mutation of interest. Mitotic recombination results in 3 types of cell populations: All cell populations will have a copy of hs flp and GAL4 driver
a) Cells with two copies of Tubp-GAL80 – GAL4 is bound by GAL80 and lacks UAS-GFP and mutation of interest – so not of our interest as cells have two wild type versions of our gene under study. (see Figure 1 )
b) Cells with one copy Tubp-GAL80 and one copy of UAS-GFP and mutataion of interest – GAL4 is bound by GAL80 – GAL4 cannot activate GFP expression and these cells are heterozygous for the mutation of interest- So again these cells are also not of our interest as these cells have one wild type copy of mutant gene. ( Not shown in figure)
c) Cells with two copies of UAS-GFP and mutation of interest (see Figure 1)- These cells don’t have GAL80 and this allows GAL4 to bind to UAS sites and activates GFP. Patches of cells which are marked positively by GFP expression also lacks the gene of interest completely- So these are the cells of interest, which can be tested for cell fate changes and possible genetic interactors of gene under study by performing antibody staining ( if antibodies against candidate genes are available).
2. One more clever way of using temperature to the advantage of researchers is by employing temperature sensitive allele of GAL80 (GAL80ts ) in combination with conventional UAS-GAl4 system ( Fig. A).
The GAL80ts results from a single glycine to arginine substitution at amino acid 203 of GAL80. The GAL80ts fails to bind GAL4 at restrictive temperatures above 29°C but binds efficiently at the permissive temperature of 18°C (Fig. B) .
We can control the activity of GAL80ts by shifting temperature thereby providing temporal control of GAL4-dependent transgene expression. This setup allows the researcher to turn GAL4 activity from “ON” mode to “OFF” by just mere shifting of temperature from 19°C to 29°C.
The GAL4 activity in presence of GAL80ts allele shows a gradual increase when we move from 18°C to 29°C. However the activity of GAL4 decreases at a particular temperature in presence of GAL80ts when compared to its levels at a similar temperature in GAL80 minus cells.
So its not completely a ON or OFF situation for GAL4 activity in presence of GAL80ts allele but a constant increase in GAL4 protein expression when we move from 18°C to 29°C.