Name: using the gal4-uas system for functional genetics in anopheles gambiae
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We gratefully acknowledge funding from LSTM and IVCC (Adriana Adolfi), BBSRC (New Investigator Award (AL), MRC (PhD studentship to BCP:MR/P016197/1), Wellcome (Sir Henry Wellcome Postdoctoral fellowship to LG: 215894/Z/19/Z) that have incorporated Gal4UAS analysis in the proposals.

1.&#160;Design and construction of UAS constructs
<ol>
	<li>Design and assembly of vectors for candidate gene expression
	<ol>
		<li>Determine the sequence to be used for candidate gene upregulation.
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			<li>Sequence the cDNA/gDNA from the strain of interest and compare it to the published sequence to verify its identity and identify potential SNPs and restriction sites for diagnostic digest.</li>
			<li>Ensure that the forward primer used for gene amplification covers the native...

<ol>
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Understanding mosquito gene function is vital to develop new approaches to control&#160;Anopheles&#160;and impact malaria transmission. The GAL4-UAS system described is a versatile and powerful system for functional analysis of candidate genes and to date we have used the system to examine the genetic basis of insecticide resistance17&#160;and cuticular hydrocarbon production15,23, as well as to fluorescently tag different mosquit...

The bipartite GAL4-UAS system is the workhorse of functional characterization of genes in the insect model organism Drosophila melanogaster1,2,3. To use the GAL4-UAS system, transgenic driver lines, expressing the yeast transcription factor GAL4 under control of a regulatory sequence, are crossed with responder lines carrying a gene of interest or RNA interference (RNAi) construct controlled by an Upstream Activation Sequence (UAS) recognized by GAL4. The progeny of this cross express the transgene of interest in a spatiotemporal pattern dictated by the promoter controlling GAL4 expression (Figure 1). Phenotypes displayed by progeny of driver-responder crosses can be assessed to elucidate the function of candidate genes. Although D. melanogaster has been used to examine genes from other organisms4,5,6,7, the GAL4-UAS system has now been adapted for use in insects of medical and agricultural importance to provide direct analysis in the species of interest 8,9,10,11,12,13,14.
In the African malaria mosquito, Anopheles&#160;gambiae, the GAL4-UAS system was first tested by cell line co-transfection9. Multiple constructs were assayed for efficiency in different pairwise combinations and found that 14 tandemly repeated UAS supplemented with a small artificial intron (UAS-14i) displayed the widest range of activation potential when used with a panel of GAL4 drivers. To demonstrate in vivo functionality, these constructs were then used to create two separate transgenic An. gambiae lines by PiggyBac transformation8: a driver line carrying GAL4 driven by a midgut specific promoter, and a responder line containing both the luciferase and enhanced yellow fluorescent protein (eYFP) genes under regulation of UAS sequences. Gut specific luciferase activity and fluorescence in the progeny indicated that the system was efficient in Anopheles. Since then, driver lines have been created expressing transgenes in other tissues important for vectorial capacity and insecticide resistance, including oenocytes15 and hemocytes16, and in a close to ubiquitous pattern10. Numerous UAS lines have also been generated to assay genes thought to be involved in metabolism and sequestration mediated insecticide resistance, cuticular hydrocarbon synthesis and to fluorescently tag different cell and tissue types (Table 1). For the responder lines, site-directed integration of the transgene is now performed by &#934;C31 catalyzed recombination cassette exchange17,18 to fix the genomic context of the UAS regulated genes. In this way, transgene expression is normalized regarding genomic insertion location, allowing for more accurate comparison of the phenotypic effects of different candidate genes.
The responder lines created to date are designed to either express the transgene either at elevated levels or to reduce gene expression through RNA interference (RNAi). Usually cDNA clones are fused to the UAS sequence to generate suitable expression plasmids, however full genomic sequences are also feasible assuming that they are not too large for cloning. To generate silencing constructs, we have used three different methods to obtain suitable tandem inverted sequences that form hairpin dsRNA that stimulates RNAi. These have included fusion PCR, asymmetric PCR and commercial synthesis of hairpin constructs. Common to each method is the inclusion of an intron sequence between the inverted&#160;sequences to provide cloning stability. Responder plasmids into which a gene of interest/RNAi construct can be inserted have been developed15. These plasmids also carry the required &#934;C31 attB sites for RMCE (described in Adolfi accompanying JoVE paper which describes the RCME technique in detail). Protocols covering the important steps required when selecting the sequence for insertion into one of these plasmids for overexpression are included in this manuscript. Additionally, two protocols for RNAi hairpin construct creation are described and illustrated.
When creating new lines, identification of rare transgenic individuals is crucial to breed from to establish and maintain transgenic colonies. Most importantly for the GAL4-UAS system there is a necessity to distinguish the responder and driver lines to establish crosses and identify individual progeny that carry both transgenes. This is achieved by using different dominant selectable marker genes linked to the driver and responder cassettes. Most commonly these are fluorescent marker genes that are clearly distinguishable using optical filters (e.g., eYFP, eCFP, dsRed). It is important that markers are expressed in a known and reliable spatiotemporal pattern as this makes identification of abnormalities and contamination easier. Fluorescent marker gene expression is routinely regulated by the synthetic 3xP3 promoter, which causes eye and ventral ganglia specific expression in all stages of An. gambiae development19. Fluorescent markers controlled by 3xP3 are included in all transformation plasmids described in this article. A protocol detailing the common methods used to screen fluorescent An. gambiae pupae GAL4-UAS lines is included here.
One of the key elements of the GAL4-UAS system is the necessity to cross the differentially marked driver and responder lines. To do this male and females from each line must be separated prior to mating. Adults are readily distinguishable by sight, however, for establishing genetic crosses it is sensible to separate the sexes prior to adult emergence to ensure that mating has not occurred. The general size difference between male and female An. gambiae pupae is too variable to be an efficient and dependable method of sex determination20. Instead clear morphological differences in the external genitalia provide a reliable basis for sexing in An. gambiae. In this article, we describe a dependable method for sexing An. gambiae pupae to set up appropriate crosses.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62131/62131fig1v3.jpg" /> 
Figure 1&#160;- Diagrammatic representation of process for using the bipartite GAL4-UAS System in Anopheles gambiae.&#160;(A) The major components of an example vector (pSL-attB-UAS14-gyp[3xp3-eYFP]) are depicted, detailing the available restriction sites (EcoRI, NheI, XhoI and NcoI) within the multiple cloning sites that are suitable for use to insert the hairpin construct or coding sequence for the gene of interest. The structure of the docking line is also depicted. (B) The crossing step is illustrated indicating the use of males from the driver line (carrying GAL4 driver by a promoter of interest and eCFP driven by the 3xP3 promoter) and females from the responder line (carrying the gene of interest or hairpin construct controlled by a UAS promoter and an eYFP marker controlled by the 3xP3 promoter). (C) A diagrammatic representation of GAL4 driving expression of the gene of interest in the progeny of the cross in B and a list of some of the typical phenotypes that are assessed. Abbreviations: Multiple Cloning Site (MCS), Recombinase mediated cassette exchange (RMCE), Upstream Activator Sequence (UAS), enhanced yellow fluorescent protein (eYFP), enhanced cyan fluorescent protein (eCFP). <a href="https://www.jove.com/files/ftp_upload/62131/62131fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
It is the use of crosses that provides the bipartite nature of the GAL4-UAS system, which has distinct advantages over more linear approaches. For example, many more combinations of driver and responder lines can be assessed than would be feasible if a new transgenic line had to be generated and maintained for each promoter/gene combination. More importantly, it allows the analysis of genes that produce lethal or sterile phenotypes when their expression is perturbed which are difficult to create/maintain in a linear system. Such lethal phenotypes can manifest at all developmental stages, depending on the gene function and spatiotemporal expression, but are most often observed during embryonic development. Visualizing mosquito embryo development requires the clearing of the opaque chorion which coats the eggs. Following methods described in Trpi&#353; (1970)21 and Kaiser et al. (2014)22, we describe the protocols we use to fix embryos, whilst maintaining structural integrity, and bleaching to clear the endochorion that allows microscopic visualization and imaging.

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	<tbody>
		<tr>
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			<td>SLS</td>
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			<td>Gilson</td>
			<td>F144059P</td>
			<td></td>
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			<td>Starlab</td>
			<td>E1412-3020</td>
			<td>Pack of 400</td>
		</tr>
		<tr>
			<td>3 mL Pasteur Pipettes</td>
			<td>SLS</td>
			<td>G612398</td>
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			<td>Fisher Scientific</td>
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			<td>Absolute Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818-500</td>
			<td>500 mL</td>
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		<tr>
			<td>Acetic Acid</td>
			<td>SLS</td>
			<td>45726-1L-F</td>
			<td>1 L</td>
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		<tr>
			<td>Cages</td>
			<td>SLS</td>
			<td>E6099</td>
			<td>30x30x30 with screen port</td>
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		<tr>
			<td>Fine Paint Brushes</td>
			<td>Amazon</td>
			<td>UKDPB66</td>
			<td>KOLAMOON 9 Pieces Detail Painting Brush Set Miniture Brushes for Watercolor, Acrylic Painting, Oil Painting (Wine Red)</td>
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			<td>Fish food</td>
			<td>Amazon</td>
			<td></td>
			<td>Tetra Min Fish Food, Complete Food for All Tropical Fish for Health, Colour and Vitality, 10 L&#160;</td>
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		<tr>
			<td>Formaldehyde Solution</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouth Aspirator</td>
			<td>John Hock</td>
			<td>612</td>
			<td></td>
		</tr>
		<tr>
			<td>Pond Salt</td>
			<td>Amazon</td>
			<td></td>
			<td>Blagdon Guardian Pond Tonic Salt, for Fish Health, Water Quality, General Tonic, pH Buffer, 9.08 kg, treats 9,092 L&#160;</td>
		</tr>
		<tr>
			<td>Pupae Pots</td>
			<td>Cater4you</td>
			<td>SP8OZ</td>
			<td>250 pots with lids</td>
		</tr>
		<tr>
			<td>Small Plastic Buckets</td>
			<td>Amazon</td>
			<td></td>
			<td>2.5 L White Plastic Pail Complete with White Lid (Pack of 10)&#160;</td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite</td>
			<td>Fisher Scientific</td>
			<td>S25552</td>
			<td></td>
		</tr>
	</tbody>
</table>

using the gal4-uas system for functional genetics in anopheles gambiae

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa.
In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

3xP3 expression of eYFP, dsRed and eCFP provides reliable, readily distinguishable identification of individuals possessing the marker genes producing expression in eyes and ventral ganglia of&#160;An. gambiae&#160;pupae (Figure 3). The differential morphology observed in male and female external genitalia used for sexing and an example of an unidentifiable pupae are highlighted in&#160;Figure 4. Removal of all water from pupae increases sexing...

Watch this Scientific Journal Video about Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae at JoVE.com

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

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.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' ...

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.

Research

JoVE Journal

Genetics

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