Current protocols最新文献

Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene-targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid-mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock-in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site-specific knock-in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samples

Basic Protocol 2: Preparation of plasmids for gene knock-in using the CRISPR/Cas9 system

Basic Protocol 3: Establishment of gene knock-in mGSC lines by electroporation gene delivery

DNA methylation is well-established as a major epigenetic mechanism that can control gene expression and is involved in both normal development and disease. Analysis of high-throughput-sequencing-based DNA methylation data is a step toward understanding the relationship between disease and phenotype. Analysis of CpG methylation at single-base resolution is routinely done by bisulfite sequencing, in which methylated Cs remain as C while unmethylated Cs are converted to U, subsequently seen as T nucleotides. Sequence reads are aligned to the reference genome using mapping tools that accept the C-T ambiguity. Then, various statistical packages are used to identify differences in methylation between (groups of) samples. We have previously developed the Differential Methylation Analysis Pipeline (DMAP) as an efficient, fast, and flexible tool for this work, both for whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS). The protocol described here includes a series of scripts that simplify the use of DMAP tools and that can accommodate the wider range of input formats now in use to perform analysis of whole-genome-scale DNA methylation sequencing data in various biological and clinical contexts. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: DMAP2 workflow for whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS)

The lung comprises multiple components including the parenchyma, airways, and visceral pleura, where each constituent displays specific material properties that together govern the whole organ's properties. The structural and mechanical complexity of the lung has historically undermined its comprehensive characterization, especially compared to other biological organs, such as the heart or bones. This knowledge void is particularly remarkable when considering that pulmonary disease is one of the leading causes of morbidity and mortality across the globe. Establishing the mechanical properties of the lung is central to formulating a baseline understanding of its operation, which can facilitate investigations of diseased states and how the lung will potentially respond to clinical interventions. Here, we present established and widely accepted experimental protocols for pulmonary material quantification, specifying how to extract, prepare, and test each type of lung constituent under planar biaxial tensile loading to investigate the mechanical properties, such as physiological stress–strain profiles, anisotropy, and viscoelasticity. These methods are presented across an array of commonly studied species (murine, rat, and porcine). Additionally, we highlight how such material properties may inform the construction of an inverse finite element model, which is central to implementing predictive computational tools for accurate disease diagnostics and optimized medical treatments. These presented methodologies are aimed at supporting research advancements in the field of pulmonary biomechanics and to help inaugurate future novel studies. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: General procedures in lung biaxial testing

Alternate Protocol 1: Parenchymal-specific preparation and loading procedures

Alternate Protocol 2: Airway-specific preparation and loading procedures

Alternate Protocol 3: Visceral pleura–specific preparation and loading procedures

Basic Protocol 2: Computational analysis

Translation of mRNA into functional proteins is a fundamental process underlying many aspects of plant growth and development. Yet, the role of translational regulation in plants across diverse tissue types, including seeds, is not well known due to the lack of methods targeting these processes. Studying the seed translatome could unveil seed-specific regulatory mechanisms, offering valuable insights for breeding efforts to enhance seed traits. Polysome profiling is a widely used technique for studying mRNAs being translated. However, the traditional method is time-consuming and has a low polysome recovery rate; therefore, it requires substantial starting material. This is particularly challenging for species or mutants with limited seed quantities. Additionally, seed polysome fractions often yield low quality RNA due to the abundance of various compounds that interfere with conventional RNA extraction protocols. Here we present a robust polysome extraction method incorporating a size-exclusion step for polysome concentration streamlined with a rapid RNA extraction method optimized for seeds. This protocol works across multiple plant species and offers increased speed and robustness, requiring less than half the amount of seed tissue and time compared to conventional methods while ensuring high polysome recovery and yield of high-quality RNA for downstream experiments. These features make this protocol an ideal tool for studying seed translation efficiency and hold broad applicability across various plant species and tissues. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Robust polysome extraction for seeds

Basic Protocol 2: Rapid fraction total RNA extraction

The platinum-based anticancer drug cisplatin and its analog carboplatin are the most used chemotherapeutic agents worldwide. It is estimated that approximately half of all cancer patients are treated with platinum drugs at some point during the therapy regimen. Cisplatin covalently binds to purine nucleobases to form DNA adducts. Cisplatin therapy is faced with two key challenges. First, despite the initial response, many patients develop cisplatin resistance. Reduced cellular accumulation of cisplatin is one common cause of therapy resistance. Second, cisplatin treatment causes general cytotoxicity, leading to severe side effects. Monitoring the subcellular concentration of platinum chemotherapeutics will help yield clinical efficacy with the minimum possible dose. Inductively coupled plasma-mass spectrometry (ICP-MS) is an analytical technique to quantify the elemental composition of various types of liquified bulk samples with high sensitivity. This article describes quantifying cisplatin accumulation in chromatin and total cell lysate using ICP-MS. The method involves treating cells with cisplatin, isolating RNA-free DNA, digesting samples, ICP-MS instrumentation, and data analysis. Although we describe these steps in one cancer cell line, the protocol can be adapted to any cell line or tissue. The protocol should be a valuable resource for investigators interested in accurate measurement of subcellular concentration of platinum and other metallo-drugs. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Cell culture conditions for A2780 cells and cisplatin treatment

Basic Protocol 2: Isolating cellular fractions and sample quantitation

Basic Protocol 3: Sample digestion, ICP-MS data collection, and analysis

Candida glabrata (Nakaseomyces glabratus) is an opportunistic fungal pathogen that has become a significant concern in clinical settings due to its increasing resistance to antifungal treatments. Understanding the genetic basis of its pathogenicity and resistance mechanisms is crucial for developing new therapeutic strategies. One powerful method of studying gene function is through targeted gene deletion. This paper outlines a comprehensive protocol for the deletion of genes in C. glabrata, encompassing primer design, preparation of electrocompetent cells, transformation, and finally confirmation of the gene deletion. The protocol begins with the identification and design of primers necessary for generating deletion constructs, involving the precise targeting of up- and downstream regions flanking the gene of interest to ensure high specificity and efficiency of homologous recombination. Followed is the preparation of electrocompetent cells, a critical step for successful transformation. Transformation of the competent cells is achieved through electroporation, facilitating the introduction of exogenous DNA into the cells. This is followed by the selection and confirmation of successfully transformed colonies. Confirmation involves the use of colony PCR to verify the correct integration of the NAT resistance cassette and deletion of the target gene. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Primer design for gene deletion in C. glabrata

Basic Protocol 2: Preparing competent C. glabrata cells

Basic Protocol 3: Transforming C. glabrata using electroporation

Basic Protocol 4: Confirming deletion strains with colony PCR

In vitro amplification of single-stranded oligonucleotide libraries presents a significant challenge due to the potential for excessive byproduct formation. This phenomenon largely affects the quality of the ssDNAs created using the most commonly used methods, e.g., asymmetric PCR, biotin-streptavidin separation, or lambda exonuclease digestion of dsDNA. Here, we describe an improved protocol that combines primer-blocked asymmetric PCR (PBA-PCR) with emulsion PCR and a cost-effective downstream process that altogether alleviates byproduct formation without distorting the sequence space of the ssDNA library. In PBA-PCR, the reaction mixture is complemented with a 3′-phosphate-blocked limiting primer that decreases mispriming, thus reducing polymerization of DNA byproducts. The downstream process includes mixing of the PBA-PCR product with excess reverse complement of the 3′-phosphate-blocked limiting primer and removal of dsDNA strands via biotin-streptavidin separation, yielding purified ssDNAs. In conclusion, we have devised a universally applicable approach for simple and cost-effective production of ssDNA libraries and unique ssDNA sequences with on-demand labeling. Our protocol could be beneficial for a variety of uses, such as generating aptamer libraries for SELEX, creating unique molecular identifiers for a wide range of sequencing applications, providing donor DNA for CRISPR-Cas9 systems, developing scaffold nanostructures, and enabling DNA-based data storage. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Amplification of ssDNA libraries using PBA-PCR

Alternate Protocol 1: Amplification of ssDNA libraries using emulsion PBA-PCR with a simplified extraction of PBA-PCR products

Basic Protocol 2: Purification of PBA-PCR products to remove dsDNA and conversion of 3′-blocked primer to double-stranded complexes

Alternate Protocol 2: Purification of PBA-PCR products to remove both dsDNA and blocking primers from the reaction mixture

Support Protocol: Analysis of PBA-PCR products by gel electrophoresis

The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Similar methods have been developed for N. meningitidis and nonpathogenic Neisseria including N. mucosa and N. musculi. Techniques are described for genetic manipulation of N. gonorrhoeae and commensal Neisseria species, as well as for growth of these fastidious organisms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Spot transformation of Neisseria gonorrhoeae on agar plates

Basic Protocol 2: Spot transformation of commensal Neisseria on agar plates

Basic Protocol 3: Transformation of Neisseria gonorrhoeae in liquid culture

Basic Protocol 4: Electroporation of Neisseria gonorrhoeae

Basic Protocol 5: Creation of unmarked mutations using a positive and negative selection cassette

Basic Protocol 6: In vitro mutagenesis of Neisseria gonorrhoeae chromosomal DNA using EZ-Tn5

Basic Protocol 7: Chemical mutagenesis

Basic Protocol 8: Complementation on the Neisseria gonorrhoeae chromosome

Alternate Protocol 1: Complementation with replicating plasmids

Alternate Protocol 2: Complementation on the Neisseria musculi or Neisseria mucosa chromosome

Basic Protocol 9: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown on solid medium

Alternate Protocol 3: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown in broth

Support Protocol: Preparing PCR templates from Neisseria gonorrhoeae colonies

Bats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size. In vivo studies of bats are challenging for several reasons, such as difficulty in locating and capturing them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation, but cells in frozen tissue are usually damaged and have low integrity and viability. Isolating primary cells from frozen tissues thus poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this is the first protocol specifically focused on fibroblast isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Bat wing biopsy collection and preservation

Support Protocol 1: Blood collection from bat venipuncture

Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy

Support Protocol 2: Primary fibroblast culture and subculture

Support Protocol 3: Determination of growth curve and doubling time

Support Protocol 4: Cell banking and thawing of primary fibroblasts

Basic Protocol 3: Lentiviral transduction of bat primary fibroblasts

Basic Protocol 4: Bat stable fibroblast cell line development

Support Protocol 5: Bat fibroblast validation by immunofluorescence staining

Basic Protocol 5: Chromosome counting

Hematoxylin and eosin staining is widely used for routine histopathological analysis under light microscopic examination to determine alterations of tissue architecture and cellular components in animal studies. Aside from hematoxylin/eosin staining, periodic acid Schiff (PAS) staining is used to detect polysaccharides and carbohydrate-rich macromolecules, and is essential in immunological fields for evaluation of glomerular lesions of kidneys in autoimmune animals. Since erythrocytes are not stained by PAS, this stain is also helpful for identifying changes in immune cells in the red pulp of the spleen, which is filled with erythrocytes. This article describes a protocol to detect Mott cells, bizarre plasma cells containing immunoglobulin inclusion bodies (Russell bodies) in the cytoplasm. The protocol can be used for formalin-fixed, paraffin-embedded tissue sections, frozen tissue sections, tissue-touch preparations, blood films, and cytocentrifuged cell smears. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Detection of Mott cells by PAS staining in formalin-fixed, paraffin-embedded tissue sections

Basic Protocol 2: Detection of Mott cells by PAS staining in frozen tissue sections, touch preparations, blood films, and cytocentrifuged cell smears