Rhizobium rhizogenes-mediated hairy root transformation has been extensively applied across various legumes, ranging from model species such as Medicago truncatula and Lotus japonicus to crop legumes like Glycine max and...Rhizobium rhizogenes-mediated hairy root transformation has been extensively applied across various legumes, ranging from model species such as Medicago truncatula and Lotus japonicus to crop legumes like Glycine max and Phaseolus vulgaris. This technique is highly regarded for its simplicity and rapid production of transgenic roots, making it invaluable for studies on gene expression and function, root biology, symbiotic interactions, and metabolic engineering. Consequently, multiple protocols have been developed to optimize its use in legumes. In this chapter, we present a detailed protocol for hairy root transformation in the model plant Lotus japonicus, emphasizing key elements essential for success that can be adapted for use in other legumes.
Sea cucumbers are highly valued for their bioactive constituents, particularly triterpenoid saponins, which exhibit diverse pharmacological properties. The complex amphiphilic nature of saponins poses significant challen...Sea cucumbers are highly valued for their bioactive constituents, particularly triterpenoid saponins, which exhibit diverse pharmacological properties. The complex amphiphilic nature of saponins poses significant challenges in their extraction and purification. Their structural diversity and amphiphilicity demand carefully optimized protocols to maximize yield and purity. Moreover, the variability of saponin profiles between different sea cucumber species and tissues necessitates reproducible methods that can be scaled from small laboratory samples to larger quantities suitable for industrial applications. This chapter describes comprehensive, reproducible protocols for both small- and large-scale extraction of saponins from sea cucumber tissues, followed by chromatographic separation and bioactivity-guided fractionation. Subsequently, analytical techniques such as thin-layer chromatography (TLC), yeast growth inhibition assays, and liquid chromatography-mass spectrometry (LC-MS) are detailed. These detailed extraction, purification, and analytical protocols facilitate downstream applications such as structural characterization, quantification, and functional studies of saponins.
Terpenoids represent one of the most structurally diverse classes of natural products, with vast potential applications in medicine, food, and agriculture. However, exploiting this potential has historically been challen...Terpenoids represent one of the most structurally diverse classes of natural products, with vast potential applications in medicine, food, and agriculture. However, exploiting this potential has historically been challenging due to their low abundance in natural sources and the economic inefficiency of synthetic chemistry approaches for such complex structures. Heterologous biosynthesis offers a promising solution to these limitations. To date, multiple heterologous hosts have been developed for terpenoid production. Among these, Nicotiana benthamiana has emerged as a prominent platform for reconstituting plant-derived natural product pathways. Its compatibility with Agrobacterium-mediated transient expression, a scalable and flexible method, enables rapid integration and functional analysis of genes from diverse plant species. Conversely, Aspergillus oryzae has gained attention as a preferred host for fungal terpenoid pathways, owing to its highly efficient fungal expression system. Herein we present methodologies for functionally validating terpenoid biosynthetic clusters in N. benthamiana and A. oryzae.
Plant specialized metabolites encompass a diverse array of bioactive compounds with significant pharmaceutical, nutraceutical, and industrial relevance, and they play vital roles in plant defense, signaling, and ecologic...Plant specialized metabolites encompass a diverse array of bioactive compounds with significant pharmaceutical, nutraceutical, and industrial relevance, and they play vital roles in plant defense, signaling, and ecological interactions. However, their complex biosynthetic pathways and low natural yields present a major bottleneck for commercial exploitation. Synthetic biology provides new tools for the rational design and engineering of metabolic pathways to enhance specialized metabolite biosynthesis in plants as well as in microbes such as E. coli and yeast. Here, we report a synthetic biology framework utilizing Golden Gate assembly (GGA) for the modular construction and optimization of plant metabolic pathways. Golden Gate enables scarless, directional assembly of multigene constructs with high efficiency, ideal for refactoring biosynthetic gene clusters and for the systematic design of complex metabolic networks. Golden Gate assembly can efficiently join up to 10-20 fragments simultaneously in a single reaction, a significant improvement over traditional molecular biology cloning methods. We developed and validated a standardized module of promoters, untranslated regions (UTRs), coding sequences, and regulatory elements compatible with plant transformation systems. Using this platform, we successfully reconstructed the biosynthetic pathway for plant specialized metabolites by transient expression in Nicotiana benthamiana, demonstrating significant improvements in construct stability. Our results highlight the potential of Golden Gate as a cornerstone technology for plant metabolic engineering, offering a scalable and versatile strategy to enhance the production of valuable specialized metabolites and set the stage for custom metabolic rewiring aimed at sustainable production of high-value compounds.
Fungi are renowned for producing a diverse range of small-molecule natural products with applications in the food, pharmaceutical, and agrochemical industries. While bioinformatics can rapidly identify potential biosynth...Fungi are renowned for producing a diverse range of small-molecule natural products with applications in the food, pharmaceutical, and agrochemical industries. While bioinformatics can rapidly identify potential biosynthetic gene clusters (BGCs), their functional analysis remains a bottleneck, limiting the discovery of novel compounds. Advances in molecular techniques and genome sequencing now enable heterologous expression of biosynthetic pathways in hosts like Aspergillus oryzae, facilitating access to metabolites not achievable through traditional methods. However, given the high number of uncharacterized gene clusters, a high-throughput process is crucial for efficiently mining novel natural products. This chapter outlines a streamlined, high-throughput protocol using yeast homologous recombination and automated liquid handling to construct multigene plasmids for heterologous expression. While this chapter uses A. oryzae NSAR1 and the pTYGS series of expression vectors as an example, the core principles of this robotics-driven protocol are adaptable to other heterologous hosts, vectors, and organisms, providing a powerful tool for high-throughput discovery and engineering of natural products.
As the cost of genome sequencing continues to decline, an increasing number of plant genomes are being sequenced and assembled. This progress allows for the comparison of synteny and microsynteny among genomes and gene c...As the cost of genome sequencing continues to decline, an increasing number of plant genomes are being sequenced and assembled. This progress allows for the comparison of synteny and microsynteny among genomes and gene clusters across various species, facilitating the exploration of their evolutionary relationships.In this chapter, we describe current methods for analyzing collinearity and synteny among multiple genomes using existing tools from JCVI. We also present a detailed case study that illustrates these methods, utilizing data from four plant genome datasets.
Multiplex Ligation-Dependent Probe Amplification (MLPA) is a robust method for efficient genotyping known structural variations, especially duplications and deletions, in a population scale. It allows for the simultaneou...Multiplex Ligation-Dependent Probe Amplification (MLPA) is a robust method for efficient genotyping known structural variations, especially duplications and deletions, in a population scale. It allows for the simultaneous assessment of multiple regions in the genome in a single assay. This feature makes MLPA especially useful in the analysis of metabolic gene clusters (MGCs), where three or more neighboring genes need to be genotyped to decipher the overall MGC structural pattern. By enabling fast and cost-effective screening of a large number of individuals or lines, MLPA may measure MGC intraspecific variation or reveal new MGC member(s), e.g., resulting from gene duplication. This in turn may provide insight into the potential diversity of biosynthetic pathways encoded by a given MGC. Currently, however, custom synthetic probes are required to target non-human genes with MLPA. Here we describe the process of designing such synthetic probes detecting MGC genes and conducting MLPA assays. MLPA with synthetic probes may be applied for MGC analysis in various species including plants, animals, or fungi.
Phytoalexins are specialized metabolites that play a crucial role in plant defense response. Similarly to other plant specialized metabolites, their biosynthetic pathways are in some cases encoded by co-localized genes t...Phytoalexins are specialized metabolites that play a crucial role in plant defense response. Similarly to other plant specialized metabolites, their biosynthetic pathways are in some cases encoded by co-localized genes that form biosynthetic gene clusters (BGCs). Discovery of BGCs responsible for phytoalexin production can significantly facilitate delineation of their biosynthesis and function. Here we describe an experimental approach for systematic identification of stress-induced BGCs, potentially involved in phytoalexin biosynthesis. Plants are treated with methyl jasmonate (MeJA) to induce stress-responsive metabolic pathways, and RNA sequencing (RNA-seq) of treated plants is applied to identify genes upregulated in response to MeJA treatment. The resulting transcriptomic data is next integrated with BGC prediction tools to identify co-localized genes, together leading to the identification of stress-induced BGC candidates. This approach provides a pipeline that serves to facilitate the discovery of novel phytoalexin biosynthetic pathways, thus providing insight into defense-related plant specialized metabolism and its underlying genetic basis.
There is increasing evidence that genes in plant genomes are not randomly distributed. Notable examples are the recently identified biosynthetic gene clusters (BGCs), which encode various specialized metabolic pathways....There is increasing evidence that genes in plant genomes are not randomly distributed. Notable examples are the recently identified biosynthetic gene clusters (BGCs), which encode various specialized metabolic pathways. The evolution of BGCs in plants is highly debated, mainly due to the complex interplay of gene duplication, cryptic genomic translocation, and selective pressures that drive their formation and diversification. We previously adopted genomic neighborhood (GN) analysis to identify and interpret evolutionary events and trajectories for BGCs specifically evolved in Brassicaceae. Here, we present step-by-step methods for conducting GN association analysis to identify significantly clustered genes, utilizing high-quality genomes from grass species as working materials. This pipeline provides a framework for understanding the organization (assessed by content similarity) and evolution (evaluated by sequence similarity) of plant BGCs, offering insights into the mechanisms underlying the distribution of genes involved in specialized metabolism.
Biosynthetic gene clusters (BGCs) are often encoding specialized metabolic pathways in plants, yet effective methods for the comparison across multiple species are still evolving. In this protocol, we present a bioinform...Biosynthetic gene clusters (BGCs) are often encoding specialized metabolic pathways in plants, yet effective methods for the comparison across multiple species are still evolving. In this protocol, we present a bioinformatics approach combining plantiSMASH and MCScan for the identification, annotation, and comparative analysis of BGCs in plant genomes. The methodology involves using plantiSMASH to predict and annotate potential BGCs, followed by the use of MCScan to perform syntenic analysis, enabling the exploration of the conservation and evolutionary dynamics of BGCs across different plant species. Here, we provide a step-by-step guide for installing and configuring the necessary software, preparing genomic data, and executing the analysis. This methodology, integrated with transcriptomic and metabolomic data, can be used to verify the functional relevance of the identified BGCs in specific biosynthetic pathways. It is applicable to a broad range of plant species and serves as a framework for the discovery and characterization of BGCs. The described methodology can significantly enhance research in plant genomics and metabolic engineering by offering new insights into the organization and function of BGCs.
Metabolic gene clusters (MGCs) are genomic loci that contain multiple genes that are functionally and genetically linked. MGCs collectively encode a spectrum of metabolic functions, including small molecule biosynthesis,...Metabolic gene clusters (MGCs) are genomic loci that contain multiple genes that are functionally and genetically linked. MGCs collectively encode a spectrum of metabolic functions, including small molecule biosynthesis, nutrient assimilation, metabolite degradation, and production of proteins essential for growth and development. Due to their diverse ecological functions, identifying gene clusters is a powerful tool for small molecule discovery and provides insight into the ecology and evolution of organisms. Gene cluster detection algorithms have historically been specialized for detecting biosynthetic gene clusters that contain canonical "core" biosynthetic functions, while overlooking uncommon or unknown cluster classes. These overlooked clusters are a potential source of novel natural products and comprise an untold portion of overall gene cluster repertoires. Unbiased, function-agnostic detection algorithms therefore provide an opportunity to reveal novel classes of gene clusters and more precisely define genome organization.We developed CLOCI (Co-occurrence Locus and Orthologous Cluster Identifier) as a generalized, unbiased gene cluster detection algorithm. CLOCI generalizes gene cluster detection by identifying signatures of coordinated gene evolution that underlie all classes of MGCs. CLOCI first detects selection on gene colocalization by identifying and circumscribing shared synteny loci across a dataset of genomes into homologous locus groups. Gene clusters comprise a subset of these homologous locus groups, and CLOCI implements orthogonal proxies of coordinated gene evolution, such as quantifying loss and horizontal transfer of a locus, to enrich MGCs from homologous loci. Here, we describe the conceptual framework of the CLOCI algorithm and present a description of its implementation (see Note 1).
Stem cell-derived exosomes have gained increasing attention due to their therapeutic potential in various diseases. The successful acquisition of exosomes with the desired therapeutic efficacy requires both the appropria...Stem cell-derived exosomes have gained increasing attention due to their therapeutic potential in various diseases. The successful acquisition of exosomes with the desired therapeutic efficacy requires both the appropriate cultivation of the source stem cells and the use of suitable isolation methods. In this context, serum-free culture approaches have emerged as a key strategy to enhance exosome yield. This chapter explains in detail how to properly grow stem cells, how to adapt them to serum-free culture conditions, and how to get exosomes from these cells.
T cell-mediated therapies are major breakthroughs in the treatment of melanoma. However, clinical hurdles in the application of T cell-mediated therapies in melanoma persist and limit their potential. As such, preclinica...T cell-mediated therapies are major breakthroughs in the treatment of melanoma. However, clinical hurdles in the application of T cell-mediated therapies in melanoma persist and limit their potential. As such, preclinical research efforts remain focused on their development and optimization. Preclinical research techniques include in vivo evaluation with xenograft mouse models, ex vivo patient tumor explant culture, and in vitro tumor spheroids and melanoma/T cell co-cultures. However, these models lack the appropriate tissue context of melanoma skin cancer. Here, we describe a technique for the incorporation of primary T cells and chimeric antigen receptor (CAR)-T cells with melanoma cells in human skin organoids derived from primary epidermal keratinocytes.
This chapter presents a stepwise optimization of a Matrigel-based in vitro angiogenesis assay using human umbilical vein endothelial cells (HUVECs) to achieve reproducible and quantifiable tube formation. Critical parame...This chapter presents a stepwise optimization of a Matrigel-based in vitro angiogenesis assay using human umbilical vein endothelial cells (HUVECs) to achieve reproducible and quantifiable tube formation. Critical parameters-including culture surface, matrix composition and thickness, coating temperature and incubation time, growth factor-defined serum-free medium, and cell seeding density-were systematically evaluated. Optimal conditions were identified as 3 mg/mL Matrigel (100 μL/well), polymerized at 22 °C for 90 min, with HUVECs seeded at 20,000 cells/cm and cultured in a defined growth factor cocktail. Standardized imaging and ImageJ-based analysis enabled robust quantification of network features. This protocol improves consistency, stability, and comparability across angiogenesis studies.
Mechanical properties play a critical role in regulating ovarian function, yet their spatial variation within intact tissue remains poorly understood due to the limitations of conventional mechanical testing techniques....Mechanical properties play a critical role in regulating ovarian function, yet their spatial variation within intact tissue remains poorly understood due to the limitations of conventional mechanical testing techniques. Confocal Brillouin microscopy offers a non-contact and label-free approach for mapping mechanical properties of the intact ovarian tissue without perturbing tissue structure. In fresh ovarian tissue, Brillouin images reveal spatial heterogeneity in mechanical properties across follicles, surrounding stroma, and fluid-filled regions, suggesting underlying differences in cellular organization and extracellular matrix composition. These measurements capture both inter- and intra-follicular variations, providing insight into regional differences in mechanical properties within and between follicles. This chapter outlines the principles of Brillouin microscopy and its application to fresh ovarian tissue, highlighting its capability to resolve microscale mechanical variations in situ. This method provides a foundation for understanding the role of mechanical properties in ovarian function, with potential applications in disease models and reproductive health research.
Studies on genome spatial organization have increased considerably in the past few decades, allowing for the detailed characterization of diverse genomic topological landscapes. Chromatin interactions between promoters a...Studies on genome spatial organization have increased considerably in the past few decades, allowing for the detailed characterization of diverse genomic topological landscapes. Chromatin interactions between promoters and regulatory elements are crucial in controlling gene regulation during development, resulting in specific gene expression profiles characterized by transcript dosage and their distribution in time and space. Here, we describe both proximity-ligation-based and non-proximity-ligation-based new technologies, as well as their exciting findings in pluripotent cells and early embryogenesis. We also discuss examples of chromatin conformation disruption in disease and cancer.
Changes in cell fate during early mammalian development are supported by the dynamic regulation of energy metabolic pathways. These transitions fulfill energetic demands and influence transcriptional reprogramming and ce...Changes in cell fate during early mammalian development are supported by the dynamic regulation of energy metabolic pathways. These transitions fulfill energetic demands and influence transcriptional reprogramming and cell differentiation. Variations in NAD+/NADH ratios during early developmental transitions correlate with metabolic remodeling. Indeed, NAD+ bioavailability acts as an early signal for a change in the metabolic landscape by controlling the redox state and activity of multiple NAD+ dependent enzymes. Here, we summarize recent studies on NAD+ bioavailability and its function as the master organizer of energy metabolism during early mouse development. Additionally, we examine the function of NAD+ dependent chromatin remodelers in synchronizing transcriptional programs with pathways that fulfil the metabolic demands of developmental stage transitions.
Many cellular processes are governed by protein-protein interactions. To better understand these processes, it is necessary to understand how different proteins interact under different conditions. Proximity ligation ass...Many cellular processes are governed by protein-protein interactions. To better understand these processes, it is necessary to understand how different proteins interact under different conditions. Proximity ligation assay (PLA) enables identification of proteins within 40 nm of one another, suggesting interaction, while also providing information on sub-cellular localization of the interaction. This information is particularly informative when monitoring the localization and interaction of transcription factors and other nuclear proteins. Here, we describe an optimized protocol for PLA in mouse embryonic stem cells, trophoblast stem cells, and blastocysts, alongside a demonstration of a robust positive control for nuclear proteins.
Flow cytometry and fluorescence-activated cell sorting (FACS) techniques have significantly advanced the characterization of adipocyte precursor cell (APC) populations. They allow immuno-phenotyping, quantification, and...Flow cytometry and fluorescence-activated cell sorting (FACS) techniques have significantly advanced the characterization of adipocyte precursor cell (APC) populations. They allow immuno-phenotyping, quantification, and isolation of distinct populations, which critical for understanding adipose tissue development and homeostasis. Here, we describe the identification and purification of APCs using flow cytometry, FACS, and cytometry by time-of-flight (CyTOF), defined by previously established surface marker profiles. In addition, we describe the mouse models and whole adipose tissue visualization techniques that will enable us to characterize the cellular origin and plasticity of APCs.
Skeletal muscle is composed of different myofiber types with specific transcriptomic, proteomic, and metabolic identities. As such, skeletal muscle represents a valuable model to study cell differentiation and gene expre...Skeletal muscle is composed of different myofiber types with specific transcriptomic, proteomic, and metabolic identities. As such, skeletal muscle represents a valuable model to study cell differentiation and gene expression regulation. However, many molecular analyses of skeletal muscle are performed on whole muscle, with the caveat that many different cell types that constitute this organ contribute to the experimental results. Here, we describe a method to efficiently isolate individual myofibers from two muscle types and provide a guide to perform quantitative gene expression analysis of representative myosin heavy chain genes.