Methods in molecular biology最新文献

To develop effective malaria transmission-blocking vaccines and drugs, it is crucial to understand the genetic factors and molecular mechanisms that regulate the development of Plasmodium blood-stage sexual forms, known as gametocytes-parasite stage capable of surviving in the mosquito vector. We established a scalable forward genetic screen approach using single-insertion mutants generated by random piggyBac mutagenesis. This method identifies genes essential for asexual parasite forms survival or tolerance to critical in vivo phenotype responses, such as febrile temperature, antimalarial drugs, and oxidative stress. Building on this well-established approach, we developed a screen for gametocyte-related phenotypes, categorizing genes based on their impact on gametocyte production and development as either hypo-producers (reduced gametocyte production) or hyper-producers (increased gametocyte production). This approach identifies the genetic factors driving gametocyte conversion and growth. Here, we present the methodology of our large-scale phenotypic screen for identifying essential Plasmodium falciparum gametocyte genes.

Understanding the dynamic changes in the intracellular metabolism of immune cells has become fundamental to understanding the regulation of their effector functions. Optical metabolic imaging, consisting of optical redox ratio and fluorescence lifetime imaging microscopy of endogenous coenzymes NAD(P)H and FAD, offers a label-free and non-invasive approach to assess intracellular metabolism at the single-cell level. The major advantage of optical metabolic imaging is that it can assess heterogeneity in the sample with spatiotemporal resolution. While this approach has been mainly used to perform metabolic imaging on in vitro samples, studies have demonstrated that it also performs well in live, intact animals, and is sensitive to dynamic changes in immune cell activation. This chapter describes protocols for performing optical metabolic imaging of innate immune cells at the caudal fin wound microenvironment of larval zebrafish following sterile injuries. However, the protocol can be readily applied to other cell types and in different biological contexts.

Microelectrode arrays offer an exciting opportunity to probe and characterize the functional status of dystrophin-deficient muscle tissue vis-à-vis patient-specific iPSC-derived skeletal muscle (SkM) and cardiac muscle (CM). Here, we present a method to culture and analyze baseline electrophysiological profiles of Duchenne muscular dystrophy (DMD) iPSC-SkM and iPSC-CM using MEA dishes.

Whole-mount X-gal staining is a classical histochemical method for detecting β-galactosidase (LacZ) expression in fixed tissues, providing spatial resolution of gene activity in situ. In cancer research, LacZ serves as a versatile reporter for monitoring gene activation, tracing cell lineage in genetically engineered models, and assessing cellular responses to oncogenic signaling within the tumor microenvironment. Here, we present an optimized protocol for the in situ visualization of LacZ⁺ cells in the skin of Ptch1^+/- SKH-1 mice-a genetically defined model of basal cell carcinoma (BCC) characterized by constitutive Hedgehog (Hh) pathway activation. In this model, LacZ expression faithfully reports Hh signaling and allows direct visualization of emerging and established BCC lesions. Importantly, our protocol preserves tissue integrity and antigenicity, enabling seamless integration with downstream immunostaining or multispectral immunofluorescence. When combined with immune markers-including those for regulatory T cells, cytotoxic T lymphocytes, myeloid subsets, and cytokine expression-this approach permits high-resolution spatial profiling of immune architecture in relation to LacZ⁺ tumor foci. This method is particularly suited for studying how oncogenic signaling pathways such as Hh shape the immune landscape during tumor initiation, progression, or therapeutic response. Overall, the protocol offers a versatile platform for coupling gene expression mapping with immune contexture analysis in preclinical models of skin cancer.

Enteric glia play fundamental roles in the plasticity and homeostasis of the enteric nervous system. Disruptions in their function contribute to intestinal dysfunction and are associated with gut pathophysiology. To further understand the role of enteric glia in gut physiology, it is essential to use a pure and reliable primary culture method. Here, we describe a protocol for the isolation, purification, enrichment, and culture of enteric glia from mature rodent intestines.

Periodontal disease (PD) is a chronic inflammatory process of infectious etiology that affects the periodontal tissues. PD is caused by the subgingival biofilm, which, in dysbiosis, leads to an uncontrolled response of the immunological system in the periodontal tissues. To further understand the mechanisms involved in PD and how it is linked to other diseases, several animal models have been developed. These models allow researchers to study the different aspects of PD in a controlled setting, such as its pathogenesis and treatment options. Oral inoculation of periodontal bacteria, such as Aggregatibacter actinomycetemcomitans or Porphyromonas gingivalis, is one of the most commonly used models for studying PD. In these methods, the bacteria are inoculated directly into the oral cavity, allowing for rapid colonization and development of the disease. Another widely used mouse model for PD involves the application of a silk ligature around the second molar, the ligature triggers oral micro-organisms accumulation inducing an inflammatory response in the surrounding tissues, leading to gingival inflammation and pocket formation. The application of mouse models of PD has several advantages, such as relatively low cost, fast results, and the possibility of performing more accurate studies. In this chapter, we will describe bacteria- and ligature-induced periodontal disease models in detailed steps.

Nucleotide sequence analyses provide insights into changes that might have an impact on proteins and their function. With the rapid accumulation of sequence data, it is now possible to recover the evolutionary history of most genes at the population level to species and beyond. Those sequences can be compared for substitutions that might change or not change the encoded protein and its function, but they can also help to estimate evolutionary relationships. These hypotheses, as phylogenetic trees, provide visual and statistical guidance for characterizing the degree of relatedness among biological entities. In a phylogenetic tree, ancestor-descendant relationships are represented by connections, and closely related entities share most of these links, which represent their evolutionary closeness. In this chapter, I outlined a method to retrieve and label nucleotide sequences of the cytokine IL17A gene, align them to identify substitutions in homologous sites, estimate phylogenetic trees with support values, and visualize these trees as images. The methodology outlined here uses free software packages in the R environment and the Python language.

The whole genome amplification (WGA) allows new clinical applications with minimal genetic material, such as in the genetic diagnosis of Mendelian diseases in embryos before implantation (Preimplantation Genetic Test for Mendelian Abnormalities, PGT-M). This approach allows couples to avoid the transmission of Mendelian disease by undergoing assisted reproduction treatment through in vitro fertilization (IVF). First, Preimplantation Genetic Testing for Aneuploidy (PGT-A) is used to identify chromosomal aneuploidies in IVF-generated embryos. Then, or in parallel, euploid embryos can be screened for specific diseases caused by variants in a single gene to achieve the conception of offspring free of a specific monogenic disease.Here, we detail the WGA preparation and two downstream usages: (1) preparation of PCR fragments for Sanger sequencing, exemplifying the diseases we detected for healthy embryo selection and transfer in IVF, and (2) detection of chromosome Y for embryo sex diagnosis.

Single-cell RNA sequencing is widely used in developmental biology, immunology, cancer research, and clinical applications, providing a scalable and reliable approach for single-cell transcriptomics. The Chromium Next GEM Single Cell 3' Reagent kits by 10× Genomics provide an advanced method for generating single-cell gene expression libraries using microfluidic partitioning and barcoding technology. These kits enable the profiling of thousands of individual cells in a single experiment by encapsulating single cells with uniquely barcoded Gel Beads-in-Emulsion (GEMs). Reverse transcription (RT) occurs within each GEM, producing barcoded cDNA, which is subsequently purified, amplified, and converted into a dual-indexed sequencing library. The workflow consists of four major steps: GEM generation and barcoding, post-GEM-RT cleanup and cDNA amplification, 3' gene expression library construction, and sequencing. Quality control measures, including SPRIselect bead cleanup, Bioanalyzer/TapeStation validation, and PCR optimization, ensure high-quality sequencing results. The final library is compatible with Illumina sequencing platforms, allowing researchers to analyze cellular heterogeneity, gene expression dynamics, and rare cell populations. This protocol is based on the 10× Chromium Single Cell 3' Reagent Kits user guide (v3.1-Dual Index), which can be downloaded from the 10× Genomics website ( https://www.10xgenomics.com ). It is recommended to refer to the original official guide for more details when carrying out the experiments with these kits, ensuring you stay updated with any revisions or updates.

In the clarification of sexual assault cases, forensic DNA profiling of single sperm cells can be desirable if only a few sperm cells are present in intimate swabs analyzed, or if sperm cells from more than one individual are present. However, the DNA content of a single sperm cell is far below the sensitivity limits of current standard forensic methods and thus does not allow for reliable DNA profiling. Here, we describe a protocol for micromanipulation of single sperm cells that is based on using an adhesive-coated tungsten needle to pick individual cells that have been spread on an agarose plate. Forensic DNA typing is thereafter accomplished by subjecting the extracted DNA to multiple strand displacement amplification (MDA) prior to analysis. For MDA, the QIAGEN REPLI-g Single Cell kit is used, and DNA profiles are analyzed using the Promega PowerPlex ESX 17 kit followed by capillary electrophoresis. Using this protocol, over 80% complete haploid DNA profiles can be obtained from the majority of single sperm cells picked.