JoVE Neuroscience is a multidisciplinary section devoted to investigations of the structure, function, physiology, and pathophysiology of the brain and nervous system. Included methodologies range from molecular and cellular level studies to full central and peripheral neural systems. Potential treatment platforms and surgical techniques for neurological diseases and disorders are also presented in this section.

Neuroscience

JoVE Medicine serves as a connection between basic research and clinical applications. Articles may present medical procedures, case studies or clinical trial methodologies. Included studies are often modeled towards predictive, preventative, personalized, and practical approaches to treatment of disease. The goal of research methodologies in this section is to improve overall patient care and prognosis.

Medicine

JoVE Immunology and Infection includes content from a variety of fields working toward a shared initiative of improving global health. Techniques evaluating the biological response to pathogens from the molecular to organismal level are presented as well as therapeutic agents and their efficacy in treating disease.

Immunology and Infection

JoVE Genetics publishes video articles demonstrating experimental approaches to investigate gene function and regulation. This includes methods to study gene expression, mutagenesis, epigenetics, genetic determinants of development, genetic disorders, population genetics and evolution.

Genetics

JoVE Environment is dedicated to research methodologies that address environmental concerns and seek to better understand Earth's ecosystem.  Special consideration is given to experimental methodologies that assess society’s impact on the environment, suggest solutions for protecting Earth’s resources, and develop sustainable fuel sources. Moving towards an environmentally conscious planet is a collaborative effort with contributions from both life and physical scientists.

Environment

JoVE Engineering encompasses a broad range of experimental and instrumental techniques utilized in physics research. Investigations in this area strive to address and answer a broad range of scientific questions, such as device mechanisms and efficiencies, using physical tools. This approach often requires a combination of specialties, and research in this area tends to be interdisciplinary with contributions from mechanical, electrical, and chemical engineers.

Engineering

JoVE Developmental Biology publishes research methodologies in the field of developmental biology. This includes methods that are used to study biological development in vitro and in vivo at the molecular, cellular, tissue, organ and whole organism levels.

Developmental Biology

JoVE Chemistry surveys a broad scope of chemical disciplines. This collection includes fundamental and applied research that investigates molecular interactions, properties, and structure spanning in the following core divisions: analytical, organic, organometallic, inorganic, bioinorganic, and biochemistry. This section also includes the design and preparation of materials and small molecules for advanced or unique applications.

Chemistry

JoVE Cancer Research illustrates experimental approaches in biomedical research and clinical practice aimed at understanding, detecting, treating and preventing cancer. The video articles in this section demonstrate methods to study the cellular and molecular mechanisms of cancer, animal models of cancer research, development of new therapies and diagnostics, tumor imaging, chemical and radiation treatments, surgical procedures and improvements in therapy. These video articles offer researchers and clinical practitioners an efficient way to learn the technologies and experimental methods in cancer research.

Cancer Research

JoVE Biology welcomes all general biology research methodologies. Content in this section canvases all fields of cell, molecular, and organismal biology, ranging from new applications of standard techniques to novel approaches aimed at understanding the functions of life and living organisms. This diverse section includes, but is not limited to, techniques in physical biology, cellular biochemistry, genetics, physiology, systems biology and a combination of eukaryotic and prokaryotic model systems.

Biology

JoVE Bioengineering merges both physical and life sciences to understand and predict biological processes. Applying physical science tools to life science questions allow for the discovery of better technologies to measure, diagnose, and clinically treat disease.

Bioengineering

JoVE Biochemistry publishes experiments that investigate the structure, function, and interactions of biological molecules. The video articles in this section demonstrate methods to study nucleic acids, proteins, carbohydrates and lipids as well as enzyme function, protein folding, molecular interactions and biochemical reactions.

Biochemistry

JoVE Behavior documents observational and experimental techniques that seek to understand human and animal behavior. Work in this field includes analysis of genetic causes and physiological responses to internal or external stimuli as well as the implicated brain regions.

Behavior

Solanum nigrumis an orphan solanaceous relative of potato and tomato, and its fresh fruit is a nutritious and delicious berry. The whole plant of S. nigrum can be used as medicine. However, the wild S. nigrum harbors some undesirable traits. Fortunately, genetic engineering techniques, especially gene-editing technologies like CRISPR/Cas9, offer valuable opportunities to rapidly improve S. nigrum by addressing these deficiencies. In most plants, plant improvement through gene-editing technology is achieved through Agrobacterium tumefaciens-mediated genetic transformation. In this article, we have detailed the genetic transformation protocols mediated by A. tumefaciens in S. nigrum. The cotyledons and hypocotyls of S. nigrum seedlings were used as explants for genetic transformation. Because S. nigrum is resistant to kanamycin, the DsRed gene was used as a visual marker to identify transgenic plants. The genetic transformation of S. nigrum can be efficiently completed according to these operation protocols. The detection of T1 transgenic plants can be directly identified by whether they emit red fluorescence, without the need for PCR identification.

genetic-transformation-mediated-agrobacterium-tumefaciens-solanum

Genetic Transformation Mediated by Agrobacterium tumefaciens in Solanum nigrum

Solanum nigrumis an orphan solanaceous relative of potato and tomato, and its fresh fruit is a nutritious and delicious berry. The whole plant of S. ...

Mutagenesis technologies have been widely used in variation breeding, directed evolution, and protein engineering. However, most methods can only induce random genomic DNA mutations that are uncontrollable, leading to deformities or even lethal effects. Here, by leveraging the fusion of three systems: cytosine base editor (CBE), blue light-inducible p-Mag/n-Mag elements, and split T7 RNA polymerase, this outlined method incorporates a blue-light-controllable, DNA region-specific, semi-random mutagenesis system.
This system can mutate cytosine to thymine on DNAs starting from the T7 promoter and ending in the T7 terminator. Any exogenous gene downstream of the T7 promoter can be potentially mutated. These tools were also designed in two parts to make the system blue-light-controllable. The first part was created by fusing CBE with n-Mag and the N-terminal-T7 RNA polymerase gene. The second part involved fusing p-Mag with the C-terminal-T7 RNA polymerase. Under blue light, these two parts were assembled to function as a mutation generator on DNAs between the T7 promoter and the T7 terminator. Without blue light, p-Mag is detached from n-Mag, releasing the CBE from editing the DNA.
The aim of this protocol is to generate region-specific random mutations in Escherichia coli with blue light control and further validate the mutated plasmids. The system can be adapted to an in vivo evolution system where the function of the gene of interest can be selected, and the desired mutations can be collected for further study.

blue-light-inducible-semi-random-mutagenesis-on-specific-exogenous

Blue Light-inducible Semi-random Mutagenesis on Specific Exogenous DNA in Escherichia coli BL21 Strain

Mutagenesis technologies have been widely used in variation breeding, directed evolution, and protein engineering. However, most methods can only induce ...

Long non-coding RNAs (lncRNAs) represent a vast and functionally diverse class of RNA molecules, with over 100,000 predicted in the human genome. Although lncRNAs are less conserved across species compared to protein-coding genes, they play critical roles in gene regulation, chromatin interactions, and cancer progression. Their involvement in cancer makes them promising therapeutic targets. CRISPR interference (CRISPRi), utilizing catalytically inactive Cas9 fused with a transcriptional repressor such as KRAB-MeCP2, offers a precise method for targeting nuclear lncRNAs and assessing their functions. This study introduces a dual CRISPRi system using orthogonal CRISPRi technologies from Staphylococcus aureus and Streptococcus pyogenes based on dCas9-KRAB-MeCP2, optimized for combinatorial targeting of lncRNAs in human melanoma cells. The protocol facilitates combinatorial gene knockdown or synthetic lethal screening of lncRNA pairs, providing a novel tool for cancer research. By exploring synthetic lethality between lncRNAs, this approach can help identify lncRNA interactions critical for cancer cell survival, offering new therapeutic strategies. The dual system's functionality is demonstrated, highlighting its potential in identifying critical cancer-specific lncRNA interactions.

dual-crispr-interference-strategy-for-targeting-synthetic-lethal

Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions Between Non-Coding RNAs in Cancer Cells

Long non-coding RNAs (lncRNAs) represent a vast and functionally diverse class of RNA molecules, with over 100,000 predicted in the human genome. Although ...

Epigenetics refers to chemical modifications of histone proteins and DNA that can regulate the expression of genes. The human epigenome is altered dynamically during cell differentiation and aging, and many diseases are associated with aberrant epigenome patterning. Recent advances in CRISPR have led to the development of programmable tools to edit epigenetic modifications at targeted genomic loci, enabling precise rewriting of epigenetic modifications in human cells. CRISPR-based epigenome editors rely on catalytically dead Cas9 coupled with epigenetic modifiers that ultimately result in programmed repression or activation of targeted genes in mammalian genomes. Unlike traditional genome editing methods, epigenome editing does not require DNA breaks or changes in the human genome sequence and thus serves as a safer alternative to control&#160;gene expression. In this protocol, we highlight two different methods to perform dCas9-mediated epigenome editing in human cell lines using plasmid DNA transfections and nucleofection of mRNAs encoding CRISPR epigenome editors. We demonstrate programmable epigenome editing to transiently repress genes using CRISPR interference (CRISPRi) and for silencing genes durably for many weeks using CRISPRoff, a fusion of dCas9 to the KRAB domain and de novo DNA methyltransferase complex. We also provide guidance on quantitative methods to measure successful epigenome editing of target genes and key considerations on which epigenome editing tool to use, depending on experimental criteria.

crispr-epigenome-editing-human-cells-using-plasmid-dna-transfection

CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery

Epigenetics refers to chemical modifications of histone proteins and DNA that can regulate the expression of genes. The human epigenome is altered ...

Next-generation sequencing (NGS) is now recognized as a powerful tool for timely and accurate drug-resistant tuberculosis (DR-TB) diagnosis. Targeted NGS (tNGS) offers a streamlined approach by focusing on specific genes associated with drug resistance, bypassing the need for traditional culture-based methods with turnaround times ranging from weeks to months. The World Health Organization (WHO) has recommended tNGS as a valuable strategy for improving tuberculosis (TB) diagnosis to guide treatment and improve treatment outcomes, particularly in resource-limited settings. Among the WHO-recommended tNGS assays, we have selected a method that provides rapid and comprehensive drug susceptibility testing, lineage determination, and strain typing. While standardized DNA extraction methods are available, they can be time-consuming and labor-intensive. To address this challenge, we optimized a simplified, matrix-based DNA extraction protocol in combination with magnetic bead purification. This method offers a rapid and efficient approach for extracting DNA directly from decontaminated sputum sediments, enabling rapid downstream tNGS analysis. By streamlining the DNA extraction process from sputum sediment, this protocol could facilitate wider adoption of tNGS in routine clinical settings, ultimately contributing to improved patient outcomes and lending to global TB control efforts.

matrix-based-dna-extraction-for-targeted-next-generation-sequencing

Matrix-based DNA Extraction for Targeted Next-Generation Sequencing on Decontaminated Sputum Samples

Next-generation sequencing (NGS) is now recognized as a powerful tool for timely and accurate drug-resistant tuberculosis (DR-TB) diagnosis. Targeted NGS ...

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

in-vivo-monitoring-transcriptional-activity-during-metabolic

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

C. elegans have long been used as a simple and accessible model to study neuronal structure and the many functions of the nervous system. Of the 302 neurons within the adult hermaphrodite nervous system, 60 are classified as ciliated sensory neurons. These neurons are central to a number of C. elegans behaviors, including but not limited to chemo-, mechano-, and osmosensing, male mating, and dauer formation. For several decades now, members of the C. elegans community have used the red fluorescent lipophilic dye DiI to visualize a subset of the ciliated sensory neurons that are directly exposed to the external environment. This dye enters the ciliated ends of the neurons and distributes in a relatively uniform pattern throughout the dendrites, cell bodies, and axons. This simple and powerful method makes an excellent first-pass tool to identify genetic mutants that impart structural or functional defects in ciliated sensory neurons. Here, we present a streamlined version of this staining method to visualize the eight pairs of amphid and two pairs of phasmid neurons that are environmentally exposed in C. elegans. We discuss tips for using this inexpensive method for imaging cellular dye-filling patterns in anesthetized animals.

dii-dye-filling-as-simple-inexpensive-tool-to-visualize-ciliated

DiI Dye-Filling as a Simple and Inexpensive Tool to Visualize Ciliated Sensory Neurons in C. elegans

C. elegans have long been used as a simple and accessible model to study neuronal structure and the many functions of the nervous system. Of the 302 ...

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila hearts before imaging is a meticulous time-intensive process that requires advanced training and motor skills. To address these challenges, we present an innovative protocol that utilizes cryosectioning for the fluorescence imaging of Drosophila heart tissue. The protocol has been demonstrated in imaging the adult Drosophila heart but could be adapted for developmental stages. The method enhances both the efficiency and accessibility of fluorescence staining while preserving the integrity of the tissue. This protocol simplifies the process without compromising the quality of imaging, thereby reducing the dependency on technicians with highly developed training and motor skills. Specifically, we replace complex techniques, such as capillary vacuum suction, with more straightforward methods like tissue embedding. This approach allows for the visualization of cardiac structures with greater ease and reproducibility. We demonstrate the utility of this protocol by effectively detecting key cardiac markers and&#160;achieving high-resolution fluorescence and immunostaining imaging that unveils intricate details of heart morphology and cellular organization. This method provides a robust and accessible tool for researchers exploring Drosophila cardiac biology, facilitating detailed analyses of heart development, function, and disease models.

novel-efficient-method-for-drosophila-heart-fluorescence-staining

Novel And Efficient Method for Drosophila Heart Fluorescence Staining with Cryosectioning

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila ...

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences that encode rRNAs, composing regions called ribosomal DNA (rDNA) loci. Many organisms' genomes contain more than one rDNA locus distributed across different chromosomes, and rRNA may be transcribed from multiple rDNA loci or only a single locus. Changes in rRNA transcription sources often indicate ribosomal distress. However, the homogeneity of rRNA obstructs distinguishing if rRNA is transcribed from multiple or a single rDNA locus. Here, we describe a method that uses single nucleotide polymorphism-sensitive fluorescence in situ hybridization (SNP-FISH) to distinguish rRNA transcription between Drosophila melanogaster rDNA loci on the X and Y chromosomes. This method leverages locus-specific rRNA variants to enable easy detection of the source of rRNA transcription with single-cell resolution. This assay can be applied to any Drosophila cell type and may be adapted for use in other systems.

single-nucleotide-polymorphism-sensitive-fish-detection-locus

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences ...

Fluorescent Protein Tagging in Zebrafish Larvae Using CRISPR/Cas9 Knock-In

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

fluorescence-labeling-to-visualize-low-expressed-proteins-in-zebrafish

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

Journal - genetics