Tool For Functional Genomics Research Articles

Silencing of ATP Synthase β Impairs Egg Development in the Leafhopper Scaphoideus titanus, Vector of the Phytoplasma Associated with Grapevine Flavescence Dorée.

Scaphoideus titanus (Hemiptera: Cicadellidae) is the natural vector of Flavescence dorée phytoplasma, a quarantine pest of grapevine with severe impact on European viticulture. RNA interference (RNAi) machinery components are present in S. titanus transcriptome and injection of ATP synthase β dsRNAs into adults caused gene silencing, starting three days post injection (dpi) up to 20 dpi, leading to decrease cognate protein. Silencing of this gene in the closely related leafhopper Euscelidius variegatus previously showed female sterility and lack of mature eggs in ovaries. Here, alteration of developing egg morphology in S. titanus ovaries as well as overexpression of hexamerin transcript (amino acid storage protein) and cathepsin L protein (lysosome proteinase) were observed in dsATP-injected females. To evaluate RNAi-specificity, E. variegatus was used as dsRNA-receiving model-species. Different doses of two sets of dsRNA-constructs targeting distinct portions of ATP synthase β gene of both species induced silencing, lack of egg development, and female sterility in E. variegatus, indicating that off-target effects must be evaluated case by case. The effectiveness of RNAi in S. titanus provides a powerful tool for functional genomics of this non-model species and paves the way toward RNAi-based strategies to limit vector population, despite several technical and regulatory constraints that still need to be overcome to allow open field application.

High-throughput design of bacterial anti-sense RNAs using CAREng.

Short RNA (sRNA) modulation of gene expression is an increasingly popular tool for bacterial functional genomics. Antisense pairing between an sRNA and a target messenger RNA results in post-transcriptional down-regulation of a specific gene and can thus be used both for investigating individual gene function and for large-scale genetic screens. sRNAs have several advantages over knockout libraries in studies of gene function, including inducibility, the capacity to interrogate essential genes and easy portability to multiple genetic backgrounds. High-throughput, systematic design of antisense RNAs will increase the efficiency and repeatability of sRNA screens. To this end, we present CAREng, the Computer-Automated sRNA Engineer. CAREng designs antisense RNAs for all coding sequences in a given genome, while checking for potential off-targets. CAREng is available as a Python script and through a web portal (https://caren.carleton.ca). Supplementary data are available at Bioinformatics Advances online.

Genome-scale CRISPR Cas9a knockout screen reveals genes that control glioblastoma susceptibility to the alkylating agent temozolomide

ABSTRACT Glioblastoma is the most fatal of all primary human brain tumors, with a 14-month median survival. The mainstay therapy for this tumor involves temozolomide, surgery, radiotherapy, and tumor-treating electric field. Cancer resistance to commonly available chemotherapeutics remains a major challenge in glioblastoma patients receiving treatment and unfavorably impacts their overall survival and outcome. However, the lack of progress in this area could be attributed to the lack of tools to probe unbiasedly at the genome-wide level the coding and non-coding elements’ contribution on a large scale for factors that control resistance to chemotherapeutics. Understanding the mechanisms of resistance to chemotherapeutics will enable precision medicine in the treatment of cancer patients. CRISPR Cas9a has emerged as a functional genomics tool to study at the genome level the factors that control cancer resistance to drugs. Recently, we used genome-wide CRISPR-Cas9a screen to identify genes responsible for glioblastoma susceptibility to etoposide. We extended our inquiry to understanding genes that control glioblastoma response to temozolomide using the genome-scale CRISPR. This study shows that the unbiased genome-wide loss of function approach can be applied to discover genes that influence tumor resistance to chemotherapeutics and contribute to chemoresistance in glioblastoma.

An Automated High-Throughput Phenotyping System for Marchantia polymorpha.

High-throughput phenotyping (HTP) allows automation of fast and precise acquisition and analysis of digital images for the detection of key traits in real time. HTP improves characterization of the growth and development of plants in controlled environments in a nondestructive fashion. Marchantia polymorpha has emerged as a very attractive model for studying the evolution of the physiological, cellular, molecular, and developmental adaptations that enabled plants to conquer their terrestrial environments. The availability of the M. polymorpha genome in combination with a full set of functional genomic tools including genetic transformation, homologous recombination, and genome editing has allowed the inspection of its genome through forward and reverse genetics approaches. The increasing number of mutants has made it possible to perform informative genome-wide analyses to study the phenotypic consequences of gene inactivation. Here we present an HTP protocol for M. polymorpha that will aid current efforts to quantify numerous morphological parameters that can potentially reveal genotype-to-phenotype relationships and relevant connections between individual traits.

Optimization of Tobacco Rattle Virus (TRV)-Based Virus-Induced Gene Silencing (VIGS) in Tomato.

Unveiling of full genome sequence of tomato demands significant advances of tomato functional genomics. Virus-induced gene silencing (VIGS) is a well explored functional genomics tool in plant biology that exploits post transcriptional gene silencing to downregulate a desired gene. Although VIGS provides an easy and highly efficient platform to study plant gene function through reverse genetics approach, currently VIGS is more efficient in model plants like Nicotiana benthamiana, which further justifies the urgent need of a highly efficient, reliable, and reproducible VIGS protocol in crop plants such as tomato. In this chapter, we have detailed an optimized Tobacco rattle virus (TRV)-based VIGS protocol in tomato.

Virus-Induced Gene Silencing for Functional Genomics of Specialized Metabolism in Medicinal Plants.

Virus-induced gene silencing (VIGS) is a functional genomics tool to transiently downregulate the expression of target gene(s) by exploiting the plant's innate defense mechanism against invading RNA viruses. VIGS is a rapid and efficient approach to analyze the gene function, particularly, in the plants that are not amenable to stable genetic transformation. This strategy has been successfully used to decipher the function of several genes and transcription factors involved in the biosynthesis of specialized metabolites and regulation of specialized metabolism, respectively, in different medicinal and aromatic plants. Here, we describe a detailed Tobacco rattle virus (TRV)-mediated VIGS protocol for silencing of the gene encoding Phytoene desaturase (PDS) in important medicinal plants Catharanthus roseus, Calotropis gigantean, Rauwolfia serpentina, and Ocimum basilicum. Our methods allow the study of gene function within 3-4weeks after agro-inoculation, and can be an easy and efficient approach for future studies on understanding of the biosynthesis of specialized metabolites in these important medicinal plants.

Highly efficient genome editing in Xanthomonas oryzae pv.oryzae through repurposing the endogenous type I-CCRISPR-Cas system.

Efficient and modular genome editing technologies that manipulate the genome of bacterial pathogens will facilitate the study of pathogenesis mechanisms. However, such methods are yet to be established for Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight. We identified a single type I‐C CRISPR‐Cas system in the Xoo genome and leveraged this endogenous defence system for high‐efficiency genome editing in Xoo. Specifically, we developed plasmid components carrying a mini‐CRISPR array, donor DNA, and a phage‐derived recombination system to enable the efficient and programmable genome editing of precise deletions, insertions, base substitutions, and gene replacements. Furthermore, the type I‐C CRISPR‐Cas system of Xoo cleaves target DNA unidirectionally, and this can be harnessed to generate large genomic deletions up to 212 kb efficiently. Therefore, the genome‐editing strategy we have developed can serve as an excellent tool for functional genomics of Xoo, and should also be applicable to other CRISPR‐harbouring bacterial plant pathogens.

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

Silencing Specific Genes in Plants Using Virus-Induced Gene Silencing (VIGS) Vectors.

As an efficient tool for functional genomics, VIGS (virus-induced gene silencing) has been widely used in reverse and forward genetics to identify genes involved in various biology processes in many plant species. Up to now, at least 50 VIGS vectors based on RNA viruses, DNA viruses or satellites have been developed for either dicots or monocots or both. Silencing specific genes using VIGS vector involves five major steps including, first, choosing an appropriate VIGS vector for the plant; second, selecting a fragment of targeted host gene; third, cloning the fragment into viral VIGS vector; forth, inoculating and infecting the appropriate plant; and fifth, quantifying silencing effects including recording silencing phenotypes and determining silencing efficiency of the target gene. In this chapter, we introduce these steps for VIGS assay in dicots and monocots, by taking a cucumber mosaic virus-based VIGS vector for Nicotiana benthamiana and maize plants as an example. Moreover, we list available VIGS vectors for monocots.

InMut-finder: a software tool for insertion identification in mutagenesis using Nanopore long reads

BackgroundBiological mutagens (such as transposon) with sequences inserted, play a crucial role to link observed phenotype and genotype in reverse genetic studies. For this reason, accurate and efficient software tools for identifying insertion sites based on the analysis of sequencing reads are desired.ResultsWe developed a bioinformatics tool, a Finder, to identify genome-wide Insertions in Mutagenesis (named as “InMut-Finder”), based on target sequences and flanking sequences from long reads, such as Oxford Nanopore Sequencing. InMut-Finder succeeded in identify > 100 insertion sites in Medicago truncatula and soybean mutants based on sequencing reads of whole-genome DNA or enriched insertion-site DNA fragments. Insertion sites discovered by InMut-Finder were validated by PCR experiments.ConclusionInMut-Finder is a comprehensive and powerful tool for automated insertion detection from Nanopore long reads. The simplicity, efficiency, and flexibility of InMut-Finder make it a valuable tool for functional genomics and forward and reverse genetics. InMut-Finder was implemented with Perl, R, and Shell scripts, which are independent of the OS. The source code and instructions can be accessed at https://github.com/jsg200830/InMut-Finder.

California TRV-based VIGS vectors mediate gene silencing at elevated temperatures but with greater growth stunting

BackgroundTobacco rattle virus (TRV) based virus-induced gene silencing (VIGS), a widely used functional genomics tool, requires growth temperatures typically lower than those of the plant’s native environment. Enabling VIGS under native conditions in the field according to applicable safety regulations could be a revolutionary advance for ecological research.ResultsHere, we report the development of an enhanced thermal tolerant VIGS vector system based on a TRV California isolate. cDNA clones representing the whole viral genome were sequenced and used to construct separate binary plant transformation vectors for functional elements of RNA1 (6765 nt) and RNA2 (3682 nt). VIGS of target genes was induced by transient transformation of the host plant with both vectors or by treating the host plant with sap from already VIGS induced plants. In Nicotiana attenuata the silencing efficiency of the PDS (phytoene desaturase) gene was 90% at 28 °C and 78% at 30 °C. Silencing at these temperatures was more prominent and durable than silencing induced by the widely used TRV PpK20-based pBINTRA6/pTV00 system, but was associated with a viral phenotype. Differences in the suppressor protein and RNA dependent RNA polymerase sequences between the TRV California isolate and PpK20 may be the reason for their different thermal tolerance.ConclusionsThe new TRV California-based VIGS vectors induce gene silencing in Nicotiana attenuata at higher temperatures than the existing pBINTRA6/pTV00 vector system, but cause greater growth defects. The new vector system opens up an avenue to study genes functions in planta under field conditions.

Regeneration in the Segmented Annelid Capitella teleta.

The segmented worms, or annelids, are a clade within the Lophotrochozoa, one of the three bilaterian superclades. Annelids have long been models for regeneration studies due to their impressive regenerative abilities. Furthermore, the group exhibits variation in adult regeneration abilities with some species able to replace anterior segments, posterior segments, both or neither. Successful regeneration includes regrowth of complex organ systems, including the centralized nervous system, gut, musculature, nephridia and gonads. Here, regenerative capabilities of the annelid Capitella teleta are reviewed. C. teleta exhibits robust posterior regeneration and benefits from having an available sequenced genome and functional genomic tools available to study the molecular and cellular control of the regeneration response. The highly stereotypic developmental program of C. teleta provides opportunities to study adult regeneration and generate robust comparisons between development and regeneration.

Silencing of Aquaporin Homologue Accumulates Uric Acid and Decreases the Lifespan of the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae).

Simple SummaryThe use of RNA interference has become increasingly popular for investigating insect physiology, testing the functionality of insect genes and as a potential control strategy. Hemiptera include many vectors for destructive plant diseases. A major characteristic of the order of Hemiptera is feeding on the phloem sap of their plant hosts. Phloem feeders face high osmotic stress between the gut lumen and hemolymph due to the high level of sucrose in phloem sap. Targeting the osmoregulation mechanisms in Diaphorina citri Kuwayama, which transmits ‘Candidatus Liberibacter asiaticus’, the putative causal agent of Huanglongbing in citrus may lead to an effective control strategy. Herein we downregulate the expression of aquaporin, representing a major mechanism of osmoregulation, by RNA interference.The Asian citrus psyllid, Diaphorina citri Kuwayama is devastating the citrus industry worldwide. It transmits ‘Candidatus Liberibacter asiaticus’, the pathogen of Huanglongbing in citrus. RNA interference is an excellent tool for functional genomics and for screening target genes for pest control. Herein, we silenced the aquaporin (AQP) gene (DcAQP) homologue in D. citri to study its functionality and whether it could be a good target for a control strategy. AQP is an integral membrane channel protein that aids in the rapid flux of water and other small solutes that move across the lipid membrane. In Hemiptera, it is well established that AQP plays important roles in adjusting to physiological challenges including (1) regulating osmotic stress between the gut lumen and hemolymph after imbibing large quantities of a low nitrogen, sugar-rich liquid diet; (2) avoiding or preventing dehydration and desiccation; and (3) surviving at elevated temperatures. The dsRNA-DcAQP was applied twice to nymphs of the 4th and 5th instars through a soaking technique. Silencing AQP caused a significant increase in nymph mortality. Emerged adults showed malformations and a shorter lifespan. Silencing DcAQP provoked alterations in some metabolites and increased the uric acid content in emerged adults. DcAQP could be a useful target to control D. citri.

A new era in functional genomics screens.

The past 25 years of genomics research first revealed which genes are encoded by the human genome and then a detailed catalogue of human genome variation associated with many diseases. Despite this, the function of many genes and gene regulatory elements remains poorly characterized, which limits our ability to apply these insights to human disease. The advent of new CRISPR functional genomics tools allows for scalable and multiplexable characterization of genes and gene regulatory elements encoded by the human genome. These approaches promise to reveal mechanisms of gene function and regulation, and to enable exploration of how genes work together to modulate complex phenotypes.

APPLICATION OF NEXT GENERATION TECHNOLOGIES: EP.321 Revolutionizing drug discovery in genetically-defined muscle disease using single-cell and high dimensional datasets

Our vision at fulcrum therapeutics is to transform the future of patients living with genetically-defined diseases. Our approach to drug discovery avoids the pitfalls that have plagued our industry and harnesses recent technological advances that allow us to forge a new path for patients. Using the latest advances in single cell methods, we can look deeper into diseased tissue to generate powerful insights into the drivers of disease and identify new ways to re-balance the cellular composition and dysregulation of pathological microenvironments. Our fulcrumseek platform brings together the deep understanding of tissue biology elucidated from the hypothesis-generating combination of snRNA-seq and spatial transcript/proteomics studies of human samples, with the latest technologies in cellular modeling and comprehensive phenotypic screening using high-throughput RNA-seq and high content imaging. We are applying the power of machine learning together with the creative and inventive spark of human intelligence to unravel the richness of this data and revolutionize drug discovery in muscular dystrophies. Using this approach, we have characterized biopsies from dystrophic muscle to understand the cellular composition, interactions, and processes that drive muscle degeneration and loss of function. Now, we are creating a database of cellular profiles generated in response to our highly curated and annotated chemical library and functional genomics tools to identify novel targets that can modulate these drivers of disease. Here, we demonstrate that by applying this integrated target discovery approach in FSHD patient biopsies as well as patient-derived myotubes in vitro, we can rapidly rediscover previously known targets in FSHD, as well as identifying novel targets. This platform has the potential to fundamentally change the way drugs are discovered and developed for patients with devastating muscle diseases.

Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes.

The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line’s most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering.

Integrated Functional Genomic and Pharmacological Characterization of the Kinome of Myeloma Cells

Small molecule kinase Inhibitors represent the main therapeutic modality for targeting aberrant kinase signaling in cancer. The recent emergence of CRISPR/Cas9 based functional genomic tools facilitates the genome-wide assessment of cancer cell dependencies, and evaluation of the relative dependence of tumor cells on diverse types of kinases inhibited by small molecule agents with different specificities, as well as other kinases for which selective inhibitors may not be currently available. In this study, we integrated the phenotypic information from the response of multiple myeloma cell lines to small molecule kinase inhibitors (16 MM cell lines, cultured in the presence vs. absence of stromal cells, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure; targeting a total of 43 known primary oncogenic targets) vs. CRISPR-based functional data on the relative dependency of MM lines on the respective targets of these inhibitors. The cell-autonomous activity of the various inhibitors was broadly potent for Aurora, PLK, and mTORC1/2 inhibitors, consistent with the high proliferative rates of MM cell lines in vitro and the role of the corresponding genes as broad-spectrum dependencies across not only MM, but also a collection of 300+ cell lines from other neoplasias. We observed virtually no anti-MM in vitro activity for selective inhibitors of c-met, ALK, EGFR, c-kit, PDGFR, VEGFR, Syk, Src, and BTK (even in cell lines with detectable transcripts for the respective kinases):, consistent with these results, the corresponding kinases genes were not essential for in vitro survival and proliferation of MM cell lines in our CRISPR studies. We observed modest to minimal response to selective pharmacological inhibitors for PDK1, PI3K (excluding those that also inhibit mTOR), and Akt. Interestingly, at the functional genomic level, PI3KCA was a prominent dependency (and more pronounced than other PI3K isoforms) for several MM cell lines, while individual isoforms of AKT were typically not essential genes for most or all of the MM cell lines tested, suggesting potential redundancy between these isoforms or feedback loops as bypass mechanisms attenuating the effect of pharmacological inhibitors. Prominent and selective MM kinase dependencies at the functional genomic level include PIM2 (providing indirect explanation for the clinical activity observed in Phase 1 trials in MM of a pan-PIM inhibitor) and IKBKB (consistent with the role of the NFKB pathway in MM). Consistent with prior studies on small inhibitors against IGF1R, its gene is a dependency in most MM lines tested and one of the most recurrent dependencies among receptor tyrosine kinases. Both pharmacological and CRISPR-based results suggest heterogeneous responses of MM cells to loss-of-function of MEK1/2 and, more broadly to diverse members of the MAPK family. Comparison of pharmacological results obtained in the presence vs. absence of stroma, indicate heterogeneous effect of bone marrow stromal cells on MM cell response to inhibitors across different targets and cell lines, including stroma-induced resistance to the same kinase inhibitor for some cell lines vs. sensitization for others. Our results provide novel insights into the relative role of different classes of kinases in MM; the similarities and potential differences in the impact of pharmacological inhibition vs. genomic perturbation of these kinases. These integrated functional genomic and pharmacological studies of the kinome provide a framework for the potential development of inhibitors against previously understudied or underappreciated kinases; and the development of molecular or functional genomic assays that may potentially guide future efforts towards individualized administration of kinase inhibitors in MM. DisclosuresMitsiades:TEVA: Research Funding; Novartis: Research Funding; Janssen/Johnson & Johnson: Research Funding; Ono: Research Funding; Abbvie: Research Funding; Takeda: Other: Employment of family member.

Identification of Key Metabolic Pathways and Biomarkers Underlying Flowering Time of Guar (Cyamopsis tetragonoloba (L.) Taub.) via Integrated Transcriptome-Metabolome Analysis.

Guar (Cyamopsis tetragonoloba (L.) Taub.) is an annual legume crop native to India and Pakistan. Seeds of the plant serve as a source of galactomannan polysaccharide (guar gum) used in the food industry as a stabilizer (E412) and as a gelling agent in oil and gas fracturing fluids. There were several attempts to introduce this crop to countries of more northern latitudes. However, guar is a plant of a short photoperiod, therefore, its introduction, for example, to Russia is complicated by a long day length during the growing season. Breeding of new guar varieties insensitive to photoperiod slowed down due to the lack of information on functional molecular markers, which, in turn, requires information on guar genome. Modern breeding strategies, e.g., genomic predictions, benefit from integration of multi-omics approaches such as transcriptome, proteome and metabolome assays. Here we present an attempt to use transcriptome-metabolome integration to understand the genetic determination of flowering time variation among guar plants that differ in their photoperiod sensitivity. This study was performed on nine early- and six delayed-flowering guar varieties with the goal to find a connection between 63 metabolites and 1,067 differentially expressed transcripts using Shiny GAM approach. For the key biomarker of flowering in guar myo-inositol we also evaluated the KEGG biochemical pathway maps available for Arabidopsis thaliana. We found that the phosphatidylinositol signaling pathway is initiated in guar plants that are ready for flowering through the activation of the phospholipase C (PLC) gene, resulting in an exponential increase in the amount of myo-inositol in its free form observed on GC-MS chromatograms. The signaling pathway is performed by suppression of myo-inositol phosphate kinases (phosphorylation) and alternative overexpression of phosphatases (dephosphorylation). Our study suggests that metabolome and transcriptome information taken together, provide valuable information about biomarkers that can be used as a tool for marker-assisted breeding, metabolomics and functional genomics of this important legume crop.

An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.).

Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.

Minimally invasive brain injections for viral-mediated transgenesis: New tools for behavioral genetics in sticklebacks.

Behavioral genetics in non-model organisms is currently gated by technological limitations. However, with the growing availability of genome editing and functional genomic tools, complex behavioral traits such as social behavior can now be explored in diverse organisms. Here we present a minimally invasive neurosurgical procedure for a classic behavioral, ecological and evolutionary system: threespine stickleback (Gasterosteus aculeatus). Direct brain injection enables viral-mediated transgenesis and pharmaceutical delivery which bypasses the blood-brain barrier. This method is flexible, fast, and amenable to statistically powerful within-subject experimental designs, making it well-suited for use in genetically diverse animals such as those collected from natural populations. Developing this minimally invasive neurosurgical protocol required 1) refining the anesthesia process, 2) building a custom surgical rig, and 3) determining the normal recovery pattern allowing us to clearly identify warning signs of failure to thrive. Our custom-built surgical rig (publicly available) and optimized anesthetization methods resulted in high (90%) survival rates and quick behavioral recovery. Using this method, we detected changes in aggression from the overexpression of either of two different genes, arginine vasopressin (AVP) and monoamine oxidase (MAOA), in outbred animals in less than one month. We successfully used multiple promoters to drive expression, allowing for tailored expression profiles through time. In addition, we demonstrate that widely available mammalian plasmids work with this method, lowering the barrier of entry to the technique. By using repeated measures of behavior on the same fish before and after transfection, we were able to drastically reduce the necessary sample size needed to detect significant changes in behavior, making this a viable approach for examining genetic mechanisms underlying complex social behaviors.