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.

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</ol>

The authors have nothing to disclose.

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 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 ΦC31 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 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 ΦC31 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="Gene editing diagram: mosquito gene/RNAi cloning, crossing setup, phenotypic analysis process." class="xfigimg" src="/files/ftp_upload/62131/62131fig1v3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1 - Diagrammatic representation of process for using the bipartite GAL4-UAS System in Anopheles gambiae. (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 Trpiš (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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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 An. gambiae 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 Figure 4. Removal of all water from pupae increases sexing difficulty ...

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' ...

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Research

JoVE Journal

Genetics

6.0K Views.  Liverpool School of Tropical Medicine. Het bipartiete GAL4-UAS systeem is een veelzijdig hulpmiddel voor functionele genetische analyse in Anopheles gambiae door genetische modificatie van genexpressie op een spatiotemporale gecontroleerde manier. Het binaire systeem maakt flexibiliteit mogelijk in experimentele benaderingen voor fenotypische karakterisering van transgenexpressie, zelfs als ernstige fitnesskosten worden geïnduceerd. Dit systeem kan worden gebruikt voor fenotypische karakterisering van genen die mogelijk betrokken...

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

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Biology

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Neuroscience

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.

The protocols reported here illustrate three alternative ways to assess the performance of genetically-engineered mosquitoes destined for vector control in laboratory-contained small cage trials. Each protocol is tailored to the specific modification the mosquito strain bears (gene drive or non-gene drive) and the types of parameters measured.

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control ...

Microinjection Method for Anopheles gambiae Embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Gene Trapping Using Gal4 in Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional mutagenesis using fluorescent reporters to tag expression of mutated genes. Several laboratories have constructed and tested enhancer- and gene-trap vectors in zebrafish, using fluorescent proteins, Gal4- and lexA- based transcriptional activators as reporters 1-7. These vectors had two potential drawbacks: suboptimal stringency (e.g. lack of ability to differentiate between enhancer- and gene-trap events) and low mutagenicity (e.g. integrations into genes rarely produced null alleles). Gene Breaking Transposon (GBTs) were developed to address these drawbacks 8-10. We have modified one of the first GBT vectors, GBT-R15, for use with Gal4-VP16 as the primary gene trap reporter and added UAS:eGFP as the secondary reporter for direct detection of gene trap events. Application of Gal4-VP16 as the primary gene trap reporter provides two main advantages. First, it increases sensitivity for genes expressed at low expression levels. Second, it enables researchers to use gene trap lines as Gal4 drivers to direct expression of other transgenes in very specific tissues. This is especially pertinent for genes with non-essential or redundant functions, where gene trap integration may not result in overt phenotypes. The disadvantage of using Gal4-VP16 as the primary gene trap reporter is that genes coding for proteins with N-terminal signal sequences are not amenable to trapping, as the resulting Gal4-VP16 fusion proteins are unlikely to be able to enter the nucleus and activate transcription. Importantly, the use of Gal4-VP16 does not pre-select for nuclear proteins: we recovered gene trap mutations in genes encoding proteins which function in the nucleus, the cytoplasm and the plasma membrane.

This protocol describes the method of gene trap insertional mutagenesis using Gal4-VP16 as the primary reporter and GFP/RFP as secondary reporters in zebrafish. Approximately one in ten high-expressing F0 fish yield gene trap progeny co-expressing GFP and RFP. The screening procedure can be readily scaled to adapt to the size of the laboratory performing the insertional mutagenesis screen.

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional ...

Fluorescence In Situ Hybridization to Study Mosquito Spermatogenesis

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Author Spotlight: Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

Immunology and Infection

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

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

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