The bipartite GAL4-UAS system is a versatile tool for modification of gene expression in a controlled spatiotemporal manner which permits functional genetic analysis in Anopheles gambiae. The procedures described for using this system are a semi-standardized cloning strategy, sexing and screening of pupae for fluorescent protein markers and embryo fixation.
The bipartite GAL4-UAS system is a versatile tool for functional genetic analysis in Anopheles gambiae through genetic modification of gene expression in a spatiotemporal controlled manner. The binary system enables flexibility in experimental approaches for phenotypic characterization of transgene expression even if severe fitness costs are induced. This system can be used for phenotypic characterization of genes potentially involved in insecticide resistance, pathogen transmission, or those that have uses in genetic control methods.
Fraser Coleman, a research technician, will help to demonstrate this procedure Begin by collecting pupae using a three-milliliter plastic Pasteur pipet with the end cut off onto a clear flat dish suitable for use with a stereo microscope. Carefully remove most of the water around the pupae. Turn on the fluorescent bulb and allow it to warm up.
Consider the color spatiotemporal patterns of a expression and ratio of different phenotypes that are expected when screening for fluorescent marker. Select the required filter on the fluorescence stereoscope and check that there is a colored beam of light visible that is directed at the center of the stage plate. Under white light, center the pupae in the field of view and bring them into focus.
Turn on off the white light and use the fine focus to bring the area of the pupae carrying the phenotype of interest with a visible fluorescent pattern into focus. Use a fine detailing paintbrush to gently turn the pupae and move the anal paddles so that the external genitalia can be identified. Separate pupae based on distinctive external genitalia.
Males have a long tube that extrudes from the final dorsal segment approximately half the length of the anal paddles. The external genitalia of female pupae are considerably shorter and bifurcate. Separate the sexed pupae into groups of 10 or less in clear 20-milliliter tubes with water, and seal the tubes with a ball of cotton wool.
Aspirate the desired number of male and female adults from the tubes into a cage or small bucket, taking care not to damage the adults during this transfer. Maintain the cross for five to seven days before blood feeding. On the following day, eggs can be collected to study embryo development.
Assemble the oviposition chamber and carefully introduce 10 to 15 blood-fed females into it. Cover the oviposition chamber and leave for 20 minutes. Carefully detach the 50-milliliter polypropylene tube from the oviposition pot, making sure not to release the mosquitoes.
Cover the pot and allow eggs to mature to the developmental stage of interest. Pick up eggs from the pot using a fine detailing paintbrush and place them on water in a 40-square millimeter excavated glass block. Carefully remove the water from the glass block with a micropipette and cover eggs in 500 microliters of FAA.
Oscillate gently on orbital shaker at room temperature for 30 minutes. Thoroughly rinse the eggs 15 times with distilled water to remove all traces of FAA solution. Using a 1000-microliter micropipette add and then remove one milliliter of distilled water at a time taking care not to damage the eggs while doing so.
Cover the fixed eggs with one milliliter of Trpis solution and incubate at room temperature for 30 minutes. Eggs will start to develop pale patches after five minutes of incubation eventually reaching a milky white color once cleared. Rinse the eggs thoroughly 15 times with distilled water to remove all traces of Trpis solution.
The expression of fluorescent markers driven by the 3xP3 promoter is observed in the eyes and ventral ganglia of Anopheles gambiae pupae. Using different colored fluorescent markers permits the selection and screening of modified individuals carrying different GAL4 and UAS components. When working with a bipartite GAL4-UAS system, it is necessary to cross males and females from different strains to acquire progeny carrying both the GAL4 and UAS components.
The differential morphology observed in male and female external genital of pupae is used for sexing. An example of an unidentifiable pupae is highlighted here. Removal of all water from pupae, as is shown on the right, increases sexing difficulty as anal paddles obscure visualization of genitalia.
The bipartite GAL4-UAS system permits modification of gene expression in a controlled spatiotemporal manner. This can be visualized by crossing the oenocyte and ubiquitous GAL4 four expressing driver lines with the responder line UAS-mCherry. A key benefit of using a bipartite GAL4-UAS system is the ease of studying lethal phenotypes even at the embryonic stage.
To do this, it is necessary to visualize the internal morphology of the embryos. The normal morphological characteristics observed in different stages of embryonic development at 12, 24, and 36 hours post-laying can be seen following completion of the embryo clearing protocol. It is vital throughout this procedure that pupae and eggs are not allowed to desiccate.
Therefore, it's important to work quickly when removing liquids. The GAL4-UAS method has allowed us to functionally characterize genes involved in insecticide resistance and those with potential uses in gene drive control methods.
