Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues¹. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.Download video file.^{(77M, mp4)}     

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America.EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1^st-, 2^nd-, 3^rd-, 4^th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.Download video file.^{(130M, mp4)}     

Accelerated publication versus usual publication in 2 leading medical journals

Background

A number of medical journals have developed policies for accelerated publication of articles judged by the authors, the editors or the peer reviewers to be of special importance. However, the validity of these judgements is unknown. We therefore compared the importance of articles published on a “fast track” with those published in the usual way.

Methods

We identified 12 “case” articles — 6 articles from the New England Journal of Medicine that were prereleased on the journal''s Web site before publication in print and 6 “fast-tracked” articles from The Lancet. We then identified 12 “control” articles matched to the case articles according to journal, disease or procedure of focus, theme area and year of publication. Forty-two general internists rated the articles, using 10-point scales, on dimensions addressing the articles'' importance, ease of applicability and impact on health outcomes.

Results

For each dimension, the mean score for the case articles was significantly higher than the mean score for the control articles: importance to clinical practice 7.6 v. 7.1 respectively (p = 0.001), importance from a public health perspective 6.5 v. 6.0 (p < 0.001), contribution to advancement of medical knowledge 6.2 v. 5.8 (p < 0.001), ease of applicability in practice 7.0 v. 6.5 (p < 0.001), potential impact on health outcomes 6.5 v. 5.9 (p < 0.001). Despite these general findings, in 5 of the 12 matched pairs of articles the control article had a higher mean score than the case article across all the dimensions.

Interpretation

The accelerated publication practices of 2 leading medical journals targeted articles that, on average, had slightly higher importance scores than similar articles published in the usual way. However, our finding of higher importance scores for control articles in 5 of the 12 matched pairs shows that current journal practices for selecting articles for expedited publication are inconsistent.A number of medical journals have developed policies for accelerated publication of articles describing findings that are judged by the authors, the editors or the peer reviewers to be particularly important and deserving of rapid dissemination. In the case of the New England Journal of Medicine, accepted articles that have “immediate clinical implications”^1,2 are occasionally prereleased on the journal''s Web site (www.nejm.org) before their official publication date, and an early press release is issued to the media. A recent high-profile example of a prereleased article is that of the RALES study that assessed the efficacy of spironolactone for congestive heart failure.^2,3The Lancet,⁴,⁵,⁶ the British Medical Journal ⁷ and CMAJ ⁸ are other medical journals that have adopted mechanisms for occasionally accelerating the peer review and printing process for articles judged to present especially important research findings needing urgent dissemination. In these journals the expedited process is referred to as “fast-track” publication. A number of other high-profile journals, including the Journal of the American Medical Association,⁹ Science¹⁰ and Nature,¹¹ have also adopted mechanisms for expedited publication, with Nature recently prereleasing on its Web site 2 articles on the molecular biology of anthrax infections.^12,13Despite the existence, and increasing profile, of these journal publication policies, no study has formally assessed the importance, methodological quality and general visibility of articles published in an accelerated manner relative to articles published in the usual manner. In this article we address these questions by asking a group of physicians to rate the importance, quality and visibility of a selection of prereleased articles from the New England Journal of Medicine and fast-tracked articles from The Lancet.     

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis^1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification^3,4, Fluorescent Activated Cell Sorting (FACS)^5-8, and RNA binding protein based strategies⁹, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type^10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells^17,18, including da neurons at the third instar larval stage of development.Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP¹⁹ transgene under the control of either the class IV da neuron specific ppk-GAL4²⁰ driver or the pan-da neuron specific 21-7-GAL4²¹ driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.Download video file.^{(137M, mp4)}     

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly three decades ago ^1,2, so far, to the best of these authors'' knowledge, no method has been developed to allow for the in situ visualization of the bacteria in histological sections.While bacterial localization has been determined in vivo in previous studies using plate counts ³, radiometric labeled ⁴, or bioluminescent bacteria ⁵, most of these studies have only been performed at the gross organ level, with one exception ⁶. This limitation is of particular concern because E. ictaluri has a complex infection cycle ^1,7, and it has a variety of virulence factors ^8,9. The complex interaction of E. ictaluri with its host is similar in many respects to Salmonella typhi ¹⁰, which is in the same taxonomic family.Here we describe a technique allowing for the detection of bacteria using indirect immuno-histochemistry using the monoclonal Ed9 antibody described by Ainsworth et al.¹¹.Briefly, a blocking serum is applied to paraffin embedded histological sections to prevent non-specific biding. Then, the sections are incubated with the primary antibody: E. ictaluri specific monoclonal antibody Ed9. Excess antibodies are rinsed away and the FitC labeled secondary antibodies are added. After rinsing, the sections are mounted with a fluorescent specific mounting medium.This allowed for the detection of E. ictaluri in situ in histological sections of channel catfish tissues.Download video file.^{(33M, mov)}     

Monitoring equilibrium changes in RNA structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling a specific biologic role as regulator, binder or catalyst. Information about tertiary contact formation is essential to understand the function of RNA molecules. Hydroxyl radicals (•OH) are unique probes of the structure of nucleic acids due to their high reactivity and small size.¹ When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA¹ and RNA² with as fine as single nucleotide resolution. Hydroxyl radical footprinting can be used to identify the nucleotides within an intermolecular contact surface, e.g. in DNA-protein¹ and RNA-protein complexes. Equilibrium³ and kinetic⁴ transitions can be determined by conducting hydroxyl radical footprinting as a function of a solution variable or time, respectively. A key feature of footprinting is that limited exposure to the probe (e.g., ''single-hit kinetics'') results in the uniform sampling of each nucleotide of the polymer.⁵In this video article, we use the P4-P6 domain of the Tetrahymena ribozyme to illustrate RNA sample preparation and the determination of a Mg(II)-mediated folding isotherms. We describe the use of the well known hydroxyl radical footprinting protocol that requires H₂O₂ (we call this the ''peroxidative'' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O₂ (we call this the ''oxidative'' protocol). An overview of the data reduction, transformation and analysis procedures is presented.Download video file.^{(82M, mov)}     

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small ''bag'' called the spermatheca. Later on, hermaphrodites continually produce oocytes¹. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for >50% of the total cells in a typical adult worm². Therefore, isolating sperm from males is easier than from that of hermaphrodites.Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome³. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population³.Males can be easily distinguished from hermaphrodites by observing the tail morphology⁴. Hermaphrodite''s tail is pointed, whereas male tail is rounded with mating structures.Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids².Males are directly dissected on a small drop of ''Sperm Medium''. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes⁵, and Nelson⁶ previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process⁷. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.Download video file.^{(33M, flv)}     

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.Several improvements of the original Coomassie protocol¹ have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution², and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol³. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff''s colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol⁴. The novel aluminum-based staining in Kang''s study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.Here, we demonstrate application of Kang''s protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.Download video file.^{(128M, mp4)}     

Sex differences in resting hemodynamics and arterial stiffness following 4 weeks of resistance versus aerobic exercise training in individuals with pre-hypertension to stage 1 hypertension

Background

Chromosomal complement, including that provided by the sex chromosomes, influences expression of proteins and molecular signaling in every cell. However, less than 50% of the scientific studies published in 2009 using experimental animals reported sex as a biological variable. Because every cell has a sex, we conducted a literature review to determine the extent to which sex is reported as a variable in cardiovascular studies on cultured cells.

Methods

Articles from 10 cardiovascular journals with high impact factors (Circulation, J Am Coll Cardiol, Eur Heart J, Circ Res, Arterioscler Thromb Vasc Biol, Cardiovasc Res, J Mol Cell Cardiol, Am J Physiol Heart Circ Physiol, J Heart Lung Transplant and J Cardiovasc Pharmacol) and published in 2010 were searched using terms 'cultured' and 'cells' in any order to determine if the sex of those cells was reported. Studies using established cell lines were excluded.

Results

Using two separate search strategies, we found that only 25 of 90 articles (28%) and 20 of 101 articles (19.8%) reported the sex of cells. Of those reporting the sex of cells, most (68.9%; n = 31) used only male cells and none used exclusively female cells. In studies reporting the sex of cells of cardiovascular origin, 40% used vascular smooth-muscle cells, and 30% used stem/progenitor cells. In studies using cells of human origin, 35% did not report the sex of those cells. None of the studies using neonatal cardiac myocytes reported the sex of those cells.

Conclusions

The complement of sex chromosomes in cells studied in culture has the potential to affect expression of proteins and 'mechanistic' signaling pathways. Therefore, consistent with scientific excellence, editorial policies should require reporting sex of cells used in in vitro experiments.     

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells¹. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of ''cellular microbiology''². Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories^3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 2787⁵, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787ΔaidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before⁶.Download video file.^{(34M, mov)}     

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)¹ and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 2². To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB³, the arginine decarboxylase AdiA⁴, the lysine decarboxylase LdcI^{5, 6} and the ornithine decarboxylase SpeF⁷. All of these enzymes utilize pyridoxal-5''-phospate as a co-factor⁸ and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate². In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 Å, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5''-diphosphate,3''-diphosphate (ppGpp) ¹⁴. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses⁹. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth¹⁰. We have demonstrated that ppGpp is a specific inhibitor of LdcI ¹⁴. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.¹¹, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the α-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)⁵. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N''-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N′-bistrinitrophenyllysine (TNP-lysine), respectively¹¹. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.Download video file.^{(50M, mov)}     

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host''s immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.Download video file.^{(94M, mov)}     

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein¹ from yeast, which couples ATP hydrolysis to protein disaggregation^2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock^3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation^13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure2^23-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils^26,31,32.Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown^33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils^4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson''s Disease²³ as well as amyloid forms of PrP³⁹. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson''s Disease²³ and Huntington''s disease³⁸. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question^4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors^30,40-42.To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide^43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His₆-tag removal compared to a previous purification method from E. coli⁴⁷. Moreover, our protocol is more facile and convenient than two more recent protocols^26,48.Download video file.^{(43M, mov)}     

Cannabinoid formation in Cannabis sativa grafted inter-racially,and with two Humulus species

Inter-racial grafts between high and low Δ¹-THC strains of Cannabis sativa, as well as cross-grafts with two Humulus (hop) species have been effected. C. sativa strains continue to produce essentially their own characteristic mixtures of cannabinoids, with undiminished vigour, whatever part of the graft system they form. There is no evidence of transport of intermediates or factors critical to cannabinoid formation across the grafts.     

Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well^9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5'' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3'' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles^3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission^5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP^2,9.Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission⁷. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.Download video file.^{(46M, mov)}