Methods in molecular biology最新文献

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.

TNBC is an aggressive and metastatic subtype of breast cancer in which the TP53 mutation occurs frequently and is associated with particularly poor outcomes. Mutations in TP53 can disrupt the intrinsic function of the tumor suppressor as well as acquire oncogenic gain-of-function (GOF) activities. However, little is known about its oncogenic GOF mediators and functions. Targeted therapy for TNBC patients is thus one of the most urgent needs in breast cancer therapeutics, and identifying genes that have synthetic lethal interactions with mutant TP53 may be a promising approach. Sequential analysis of RNA-seq followed by high-throughput RNA interference screening (HTS-RNAi screening), as an intrinsic cellular mechanism for the identification of genes with synthetic lethality of mutant TP53, is a promising strategy for the treatment of mutant TP53 in TNBC and determining its impact on tumorigenesis.

CRISPR/Cas9-based genome editing is an inexpensive and efficient tool for genetic modification. Here, we present a methodological approach for establishing interleukin-17 receptor B (IL17RB) knockout cell lines using CRISPR/Cas9-mediated genomic deletion. The IL17RB gene encodes for a cytokine receptor that specifically binds to IL17B and IL17E and is overexpressed in various cancers. The method involves CRISPR design, CRISPR cloning, delivery of the CRISPR clone into cells, and verification of IL17RB gene deletion by deletion screening primer design, genomic DNA extraction, and polymerase chain reaction (PCR). A similar approach can be used for generating mammalian cell lines with gene knockout for other genes of interest.

Chimeric antigen receptor T cells (CAR T cells) therapy has revolutionarily changed the landscape of immunotherapy and been approved by the U. S Food and Drug Administration (FDA) since 2017 for several blood malignancies. To translate novel CAR T cells into clinical applications, it is essential to evaluate their antigen specificity, cytotoxic capacity, and off-target effects in vitro. A commonly used criteria to assess CAR T cell functionality involves detecting cytokine secretion following their engagement with target antigens. This chapter describes a method of combining intracellular cytokine staining and multi-color flow cytometry to measure CAR T cells activation following antigen stimulation.

Preimplantation genetic testing (PGT) is crucial for selecting embryos free of genetic abnormalities. However, existing PGT approaches often necessitate separate platforms for aneuploidy (PGT-A), monogenic disorders (PGT-M), and structural rearrangements (PGT-SR). This can drive up costs and operational complexity when multiple PGT tests are required for a single embryo. Here, we present a MALBAC-based method that integrates PGT-A, PGT-M, and PGT-SR into one unified platform.

TAPBPR has previously been identified as a homolog of tapasin, though the two proteins serve as mutually exclusive peptide editors. While tapasin functions solely as a constituent of the peptide loading complex, TAPBPR can function alone and independently of other chaperones and cofactors. An additional characteristic of TAPBPR is its lack of an endoplasmic-reticulum retention motif, which enables it to leak to the surface of particular cell types when overexpressed as well as an ability to promote peptide exchange at the cell surface as a recombinant soluble protein. The aforementioned features of TAPBPR provide the protein with unique capabilities for the characterization of its function, as well as the ability to dissect other properties of peptide loading such as peptide affinity for major histocompatibility complex class I and immune response to the presentation of immunoreactive peptide. Here, we describe the key methods used to decorate cells with peptides, permitting the assessment of the function of TAPBPR and its variants: peptide loading, peptide dissociation, and peptide exchange assays. The use of these assays confers the ability to dissect the catalytic function of TAPBPR and its variants, as well as conducting subsequent experiments utilizing the efficient decoration of cells with immunoreactive peptide.

Morphological profiling with the Cell Painting assay started a new era in phenotypic screening approaches by harnessing comprehensive morphological profiles of cellular perturbations. This enables an unbiased characterization of the effects of small chemical compounds. We established and extensively validated a Cell Painting protocol to screen the compound libraries of the European initiative EU-OPENSCREEN ( www.eu-openscreen.eu ) in order to find robust and reproducible links between the known target or the pathway mechanism of each compound to the phenotypic profile. In this chapter, we describe the Cell Painting procedure, image analysis, and the downstream data processing procedures.

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.

Many gene regulatory processes depend on proteins that interact with DNA. Characterizing these interactions can shed light on the molecular mechanisms that allow cells to control which RNA is made, when it is made, and how much is made. Additionally, DNA protein interactions are essential for cell division, DNA replication, and repair. Chromatin ImmunoPrecipitation followed by sequencing (ChIP-seq) has been an invaluable tool for understanding gene regulatory processes in the eukaryotic kinetoplastid parasite Trypanosoma brucei by mapping genomic binding sites for a protein of interest. We recently sought to expand the repertoire of available techniques to interrogate protein-DNA interactions by optimizing Cleavage Under Targets and Release Using Nuclease (CUT&RUN) for use in Trypanosoma brucei. The protocol presented here details a CUT&RUN protocol suitable for proteins in small complexes for which there is an available antibody.

Molecular profiling is a powerful strategy for dissecting molecular aspects of cell functioning. The precision of molecular profiling is highly dependent on purity and homogeneity of cell samples. Cell technologies need cell populations enriched with target cells. Numerous approaches have been proposed to isolate and collect homogenous populations of cells and even single cells for downstream molecular profiling and recultivation. Unlike other methods for isolation of live cells, laser microdissection allows for collecting single cells and cell colonies from adherent cell cultures without detaching cells from the substrate. This is advantageous for subsequent downstream omics analyses or recultivation because of preserving cells in their intact state. Here, we present a protocol for live cell laser microdissection with gravity transfer. This approach allows for isolating fragments of cell monolayers and single cells for downstream molecular profiling and recultivation.