Biological Sciences

Zebrafish offer numerous advantages as a vertebrate model because of their rapid development, high fecundity, transparent embryos, and ease of genetic manipulation. A wide variety of transgenic and mutant fish lines have been generated, and efficiently sharing these resources is crucial for advancing research. Zebrafish lines have typically been exchanged as early embryos, adult fish, or cryopreserved sperm, making transportation costly and logistically challenging. Here, we provide a protocol for preserving functional zebrafish sperm for more than 7 days at room temperature and subsequent in vitro fertilization using the preserved sperm. In this protocol, sperm collected either from the cloaca of an anesthetized male or from dissected testes is stored in L-15-based storage medium. Importantly, the storage medium, originally developed for zebrafish, is also applicable to medaka, another widely used vertebrate model. This sperm storage method allows researchers to ship sperm using low-cost methods and to investigate key factors for motility and fertilizing ability in those sperm.

Protein S-nitrosylation is a critical post-translational modification that regulates diverse cellular functions and signaling pathways. Although various biochemical methods have been developed to detect S-nitrosylated proteins, many suffer from limited specificity and sensitivity. Here, we describe a robust protocol that combines a modified biotin-switch technique (BST) with streptavidin-based affinity enrichment and quantitative mass spectrometry to detect and profile nitrosylated proteins in cultured cells. The method involves blocking free thiols, selective reduction of nitrosothiols, biotin labeling, enrichment of biotinylated proteins, and identification by tandem mass tag (TMT)-based quantitative mass spectrometry. Additionally, site-directed mutagenesis is employed to generate “non-nitrosylable” mutants for functional validation of specific nitrosylation sites. This protocol provides high specificity, quantitative capability, and versatility for both targeted and global analysis of protein nitrosylation.

The RNA-guided CRISPR-Cas9 endonuclease has been a transformative tool for laboratory biochemistry with huge potential as a precision therapeutic. This tool site-specifically cleaves double-stranded DNA following the recognition of a unique protospacer-adjacent motif (PAM). Activation of the protein–nucleic acid Cas complex has also been widely recognized to feature an allosteric mechanism dependent on structural remodeling and interdomain crosstalk. Biophysical methods have probed the impact of allosteric perturbations on cleavage and specificity of Cas9, with the aim of engineering enhanced Cas effectors. These studies include Cas9 from thermophilic organisms that edit at higher temperatures and are active in human plasma. Validation of biophysical insights has necessitated the quantitation of DNA cleavage in vitro and, subsequently, the adaptation of established protocols to encompass temperature-dependent function that is evident in extremophilic Cas systems, such as Cas9 from Geobacillus stearothermophilus and the mesophilic SpCas9. This protocol is advantageous for probing functional temperature ranges of DNA cleavage that can theoretically be applied to any Cas-RNP system.

Telomere shortening is a hallmark of human aging, and telomerase regulation plays a critical role in cellular proliferation and replicative senescence. In human cells, telomere length imposes a limit on proliferative potential, a phenomenon known as the Hayflick limit. However, species-specific differences in telomere dynamics and telomerase regulation between humans and mice present challenges to using mice as accurate models for human telomere-related research. To address this limitation, we engineered a humanized telomerase gene (hmTert) in mice by replacing the non-coding sequences within the mouse Tert locus (mTert) with corresponding regulatory sequences from the human TERT gene. Breeding of these genetically modified mice resulted in progressive telomere shortening over successive generations, ultimately reaching human-like lengths (below 10 kb). This protocol outlines the development of this humanized telomere mouse model, referred to as HuT mice, offering a robust platform for studying human telomere biology and aging-related diseases.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a widely used programmable nuclease system for gene modification in many organisms, including Physcomitrium patens. P. patens is a model species of moss plants, a basal land plant group, which has been extensively studied from the viewpoint of evolution and diversity of green plant lineages. So far, gene modifications by CRISPR/Cas9 in P. patens have been carried out exclusively by the polyethylene glycol (PEG)-mediated DNA transfer method, in which a transgene (or transgenes) is introduced into protoplast cells prepared from protonemal tissues. However, this PEG-mediated method requires a relatively large amount of transgene DNA (typically 30 µg for a single transformation), consists of many steps, and is time-consuming. Additionally, this PEG-mediated method has only been established in a few species of moss. In the current protocol, we succeeded in CRISPR/Cas9-induced targeted mutagenesis of P. patens genes by making good use of the biolistic method, which i) requires amounts of transgene DNA as low as 5 μg for each vector, ii) consists of fewer steps and is time-saving, and iii) is known to be applicable to a wide variety of species of plants.

CRISPR-Cas9 has democratized genome engineering due to its simplicity and efficacy. Adapted from a bacterial defense mechanism, CRISPR-Cas9 comprises the Cas9 endonuclease and a site-specific guide RNA. In vivo, the Cas9 ribonucleoprotein (RNP) can target specific genomic loci and generate double-strand breaks. Eukaryotic endogenous DNA repair mechanisms recognize the cut site and attempt to repair the DNA either by non-homologous end joining, which introduces insertions/deletions, resulting in a loss of reading frame in coding genes, or through homology-directed repair that maintains the reading frame. The latter approach allows the insertion of fluorescent reporter sequences in frame with protein-coding genes in order to monitor gene expression and protein dynamics in cells and whole organisms. Here, we provide a protocol for targeting endogenous genes to introduce sequences coding for fluorescent reporters in medaka (Oryzias latipes). The method is simple, robust, and efficient, thus facilitating straightforward organismal genome editing.

Plant growth–promoting rhizobacteria (PGPR) can be used as biofertilizers to enhance crop growth for better yield and soil fertility restoration. PGPR possesses certain traits such as nutrient solubilization, phytohormone production, and production of key enzymes for improved crop growth. These traits are also important for inhibiting the growth of plant root pathogens, improving root development, and conferring stress tolerance. However, the mere presence of PGPR traits in isolated bacteria may not directly reflect an improvement in plant growth, warranting researchers to evaluate phenotypic and physiological changes upon inoculation. The current manuscript provides a detailed step-by-step procedure for inoculating the PGPR Staphylococcus sciuri into seeds and seedlings of rice and tomato plants for visualizing the enhancement of root and shoot growth. The surface-sterilized seeds of rice and tomato plants are inoculated overnight with an actively grown log-phase culture of S. sciuri, and differences in growth and biomass of seedlings that emerged from the inoculated and uninoculated seeds are analyzed 10 days after germination. Plants grown in pots with sterile soil are also treated with PGPR S. sciuri by soil drenching. A remarkable increase in root and shoot growth is observed in inoculated plants. We suggest that treating seeds with bacteria and enriching the soil with bacterial inoculum provides an adequate load of PGPR that facilitates growth improvement. This method can be a reliable choice for screening and evaluating plant growth promotion by either isolated bacteria or bacterial consortia with plant-beneficial traits.

Inflammatory bowel disease (IBD) is highly prevalent globally and, in the majority of cases, remains asymptomatic during its initial stages. The gastrointestinal microbiota secretes volatile organic compounds (VOCs), and their composition alters in IBD. The examination of VOCs could prove beneficial in complementing diagnostic techniques to facilitate the early identification of IBD risk. In this protocol, a model of sodium dextran sulfate (DSS)-induced colitis in rats was successfully implemented for the non-invasive metabolomic assessment of different stages of inflammation. Headspace–gas chromatography–mass spectrometry (HS–GC–MS) was used as a non-invasive method for inflammation assessment at early and remission stages. The disease activity index (DAI) and histological method were employed to assess intestinal inflammation. The HS–GC–MS method demonstrated high sensitivity to intestine inflammation, confirmed by DAI and histology assay, in the acute and remission stages, identifying changes in the relative content of VOCs in stools. HS–GC–MS may be a useful and non-invasive method for IBD diagnostics and therapy effectiveness control.

Extracellular vesicles (EVs) are membrane-bound, non-replicating particles released by virtually all types of cells. EVs concentrate and deliver a plethora of biomolecules driving very important biological functions, including intercellular communication not only between cells of the same organism but also across different kingdoms. Plant extracellular vesicles (PEVs) are a promising alternative to mammalian EVs in biomedical applications. Here, we present an optimized and reproducible protocol for isolating PEVs from the hairy root (HR) cultures of medicinal plants Salvia dominica and S. sclarea. Our methodological approach introduces a significant advancement in the standardization of HR-EVs purification processes from plant biotechnological platforms, paving the way for their broader application across various sectors, including agriculture, pharmaceuticals, and nutraceuticals.

The adeno-associated virus serotype 9 (AAV9)-delivered gene expression driven by the cardiac troponin T (Tnnt2) promoter is broadly considered to be cardiac-specific. However, in cases where low AAV expression is sufficient to trigger a profound biological effect in CRISPR/Cas9 gene editing, the ectopic AAV9-Tnnt2 expression and gene editing in the liver becomes non-negligible. MicroRNA122 is a microRNA that is specifically expressed in the liver. The incorporation of the microRNA122 target sequence (miR122TS) into the 3' untranslated region (UTR) of the AAV transgene could reduce ectopic gene expression in the liver. Here, we provide a protocol for sgRNA design, plasmid construction, AAV packaging, and in vivo validation of a new AAV9-Tnnt2-SaCas9-miR122TS vector using publicly available materials and tools. The application of this new vector enables cardiac-specific gene editing while circumventing leakages in the liver.