Abstract Orsay virus infection in the nematodeCaenorhabditis eleganspresents an opportunity to study host‐virus interactions in an easily culturable, whole‐animal host. Previously, a major limitation ofC. elegansas a model for studying antiviral immunity was the lack of viruses known to naturally infect the worm. With the 2011 discovery of the Orsay virus, a naturally occurring viral pathogen,C. eleganshas emerged as a compelling model for research on antiviral defense. From the perspective of the host, the genetic tractability ofC. elegansenables mechanistic studies of antiviral immunity while the transparency of this animal allows for the observation of subcellular processes in vivo. Preparing infective virus filtrate and performing infections can be achieved with relative ease in a laboratory setting. Moreover, several tools are available to measure the outcome of infection. Here, we describe workflows for generating infective virus filtrate, achieving reproducible infection ofC. elegans, and assessing the outcome of viral infection using molecular biology approaches and immunofluorescence. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of Orsay virus filtrate Support Protocol: SynchronizeC. elegansdevelopment by bleaching Basic Protocol 2: Orsay virus infection Basic Protocol 3: Quantification of Orsay virus RNA1/RNA2 transcript levels by qRT‐PCR Basic Protocol 4: Quantification of infection rate and fluorescence in situ hybridization (FISH) fluorescence intensity Basic Protocol 5: Immunofluorescent labeling of dsRNA in virus‐infected intestinal tissue
more »
« less
The West African lungfish secretes a living cocoon during aestivation with uncertain antimicrobial function
Abstract One of the most exceptional adaptations to extreme drought is found in the sister group to tetrapods, the lungfishes (Dipnoi), which can aestivate inside a mucus cocoon for multiple years at reduced metabolic rates with complete cessation of ingestion and excretion. However, the function of the cocoon tissue is not fully understood. Here we developed a new more natural laboratory protocol for inducing aestivation in the West African lungfish,Protopterus annectens,and investigated the structure and function of the cocoon. We used electron microscopy and imaging of live tissue-stains to confirm that the inner and outer layers of the paper-thin cocoon are composed primarily of living cells. However, we also repeatedly observed extensive bacterial and fungal growth covering the cocoon and found no evidence of anti-microbial activity in vitro againstE. colifor the cocoon tissue in this species. This classroom discovery-based research, performed during a course-based undergraduate research experience course (CURE), provides a robust laboratory protocol for investigating aestivation and calls into the question the function of this bizarre vertebrate adaptation.
more »
« less
- Award ID(s):
- 1938571
- PAR ID:
- 10668865
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
ABSTRACT Undergraduate laboratory courses should teach not only core experimental skills, but also the relevance of those skills to addressing contemporary scientific challenges. In this course, entitled Biochemistry & Molecular Biology Laboratory, students clone a gene into an expression plasmid, express and purify an enzyme, and perform kinetic assays on the enzyme. The enzyme chosen for this experimental sequence is TEM‐1, a β‐lactamase enzyme that is a key mediator of resistance to β‐lactam antibiotics, such as penicillins. In parallel, the students gain experience in microbiology and natural product extractions by first isolating aStreptomycesspecies from soil and then screening this isolate for antibiotic activity and the ability to produce compounds that inhibit TEM‐1 enzymatic activity. In principle, this research effort could lead to the discovery of either a novel strain ofStreptomyces clavuligerus, which produces the important TEM‐1 inhibitor clavulanate, or anotherStreptomycesstrain producing a novel TEM‐1 inhibitor. Thus, students are introduced to a pressing problem in contemporary medicine, antibiotic resistance, while they participate in discovery‐driven efforts to solve that problem. Incorporation of discovery‐based objectives into the laboratory sequence gives students individualized datasets to analyze, thereby promoting the development of student analytical skills. In addition, during the course, the students enhance their scientific writing skills through a series of peer‐reviewed writing assignments and ultimately complete a formal journal‐style article, in which they communicate their research findings. Overall, this course compares favorably with a course‐based undergraduate research experience (CURE) course, as gauged by student‐reported learning gains and overall course assessment.more » « less
-
Macdonald, Stuart (Ed.)Abstract Gene regulatory networks specify the gene expression patterns needed for traits to develop. Differences in these networks can result in phenotypic differences between organisms. Although loss-of-function genetic screens can identify genes necessary for trait formation, gain-of-function screens can overcome genetic redundancy and identify loci whose expression is sufficient to alter trait formation. Here, we leveraged transgenic lines from the Transgenic RNAi Project at Harvard Medical school to perform both gain- and loss-of-function CRISPR/Cas9 screens for abdominal pigmentation phenotypes. We identified measurable effects on pigmentation patterns in the Drosophila melanogaster abdomen for 21 of 55 transcription factors in gain-of-function experiments and 7 of 16 tested by loss-of-function experiments. These included well-characterized pigmentation genes, such as bab1 and dsx, and transcription factors that had no known role in pigmentation, such as slp2. Finally, this screen was partially conducted by undergraduate students in a Genetics Laboratory course during the Spring semesters of 2021 and 2022. We found this screen to be a successful model for student engagement in research in an undergraduate laboratory course, that can be readily adapted to evaluate the effect of hundreds of genes on many different Drosophila traits, with minimal resources.more » « less
-
Schubert, Michael (Ed.)Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to testin vivoelectroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissue-specific cells of five species from across three families of tropical frogs: poison frogs (Dendrobatidae), cryptic forest/poison frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or tissue transparency. Specifically, we examine the effects of electrical pulse and injection parameters on species- and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increased with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extendingin vivoelectroporation to non-model amphibian species could provide new opportunities for exploring topics in genetics, behavior, and organismal biology.more » « less
-
Abstract Course‐based undergraduate research experiences (CUREs) provide students with valuable opportunities to engage in research in a classroom setting, expanding access to research opportunities for undergraduates, fostering inclusive research and learning environments, and bridging the gap between the research and education communities. While scientific practices, integral to the scientific discovery process, have been widely implemented in CUREs, there have been relatively few reports emphasizing the incorporation of core biology concepts into CURE curricula. In this study, we present a CURE that integrates core biology concepts, including genetic information flow, phenotype–genotype relationships, mutations and mutants, and structure–function relationships, within the context of mutant screening and gene loci identification. The design of this laboratory course aligns with key CURE criteria, as demonstrated by data collected through the laboratory course assessment survey (LCAS). The survey of undergraduate research experiences (SURE) demonstrates students' learning gains in both course‐directed skills and transferrable skills following their participation in the CURE. Additionally, concept survey data reflect students' self‐perceived understanding of the aforementioned core biological concepts. Given that genetic mutant screens are central to the study of gene function in biology, we anticipate that this CURE holds potential value for educators and researchers who are interested in designing and implementing a mutant screen CURE in their classrooms. This can be accomplished through independent research or by establishing partnerships between different units or institutions.more » « less
An official website of the United States government

