Methods in Molecular Biology

Short hairpin RNA (shRNA) is a technique used to silence gene expression stably in various cells. There are however several reported problems. First, the cloning of oligos can lead to ligation of multiple copies; second, premature termination of sequencing reaction during confirmation of hairpin template; third, microdeletions/substitutions in hairpin during cloning; and fourth, off target effects. In this chapter, we have described a retrovirus transduction-based protocol that can be used on cells in culture without encountering any of the reported issues...

A type of three-dimensional (3D) cell culture models which is simple and easy is hanging drop method. The hanging drop method emerges as a pivotal technique with diverse applications in cancer research and cell biology. This method facilitates the formation of multicellular spheroids, providing a unique environment for studying cell behavior dynamics. The hanging drop method's theoretical underpinning relies on gravity-enforced self-assembly, allowing for cost-effective, reproducible 3D cell cultures with controlled spheroid sizes...

Immunofluorescence, a transformative tool in cellular biology, is employed to dissect the intricate mechanisms of cholesterol trafficking in human reproductive tissues. Autophagy, a key player in cellular homeostasis, particularly lipophagy, emerges as a free cholesterol source for steroidogenesis. In this chapter, we describe a comprehensive immunofluorescence staining protocol, with details provided for the precise visualization of subcellular dynamics of mitochondria, lysosomes, and lipid droplets in ex vivo testicular tissue and primary luteal granulosa cell culture models, pivotal components in sex steroid biosynthesis...

Due to their full differentiation capacity in vitro, the culture of human primary keratinocytes (HPKs) represents a physiological model for answering basic biological and dermatological research questions, including those related to skin diseases and the investigation of treatment options. When modified with the CRISPR/Cas9 gene editing approach and cultivated in organotypic 3D epidermal equivalents (EEs), these human cells have the potential to replace established mouse models. However, even when cultivated on feeder cells, HPKs have only a low proliferation capacity in 2D culture, limiting their application potential...

The mammalian integumentary system, including skin and its appendages, serves as a protective barrier for the body. During development, skin epidermis undergoes rapid cell division and differentiation to form multiple stratified layers of keratinocytes. Concurrently the epidermis also gives rise to hair follicles that invaginate into the dermis. In adult skin, the hair follicle undergoes cyclic regeneration fueled by hair follicle stem cells located in the bulge. Three-dimensional and high-resolution imaging of these structures using whole-mount immunofluorescent staining allows to better characterize epidermal progenitors and stem cells...

Diabetic foot ulcers (DFUs) pose a significant threat to the health and well-being of individuals with diabetes, often leading to lower limb amputations. Fortunately, epidermal stem cell therapy offers hope for improving the treatment of DFUs. By leveraging 3D culture techniques, the scalability of stem cell manufacturing can be greatly enhanced. In particular, using bioactive materials and scaffolds can promote the healing potential of cells, enhance their proliferation, and facilitate their survival. Furthermore, 3D tissue-mimicking cultures can accurately replicate the complex interactions between cells and extracellular matrix, thereby ensuring that the stem cells are primed for therapeutic application...

In oncological research, the function of tumor-infiltrating natural killer (NK) cells in skin carcinoma presents a viable avenue for novel therapeutic methods. NK cells are essential to the body's defense against malignancies, including skin cancer, and are especially important in more sophisticated cancer immunotherapies such as vaccinations containing dendritic cells. The deadliest type of skin cancer, malignant melanoma, still has a poor prognosis even with advancements in early-stage therapies, which emphasizes the need for novel therapeutic strategies...

Vitiligo is a skin condition affecting 1% of the global population, causing non-scaly, chalky-white macules on the skin and hair. It is caused by the pathologic destruction of melanocytes, which produce melanin. Research has focused on the abnormalities of melanocytes and their interaction with neighboring keratinocytes. Current treatments are mainly immunosuppressive drugs and UV radiation, which are scarce and ineffective. To treat vitiligo, regenerative medicine techniques, such as cell-based and cell-free methods, are recommended...

Site-directed mutagenesis (SDM) is a technique that allows mutation of specific nucleotide(s) in a codon to study its functional implications in a protein. Commercial kits are available, which require high-performance liquid chromatography purified oligos for this purpose. These kits are expensive, and they are not very efficient, so one has to sequence several clones to get a desired one. We present here a simple method that requires only crude oligos, commercially available high-fidelity enzymes, and the success rate is close to 100%...

Single-cell RNA sequencing (scRNA-seq) has been widely applied in neuroscience research, enabling the investigation of cellular heterogeneity at the transcriptional level, the characterization of rare cell types, and the detailed analysis of the stochastic nature of gene expression. Isolation of single nerve cells in good health, especially from the adult rodent brain, is the most difficult and critical process for scRNA-seq. Here, we describe methods to optimize protease digestion of brain slices, which enable yield of millions of cells in good health from the adult brain...

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by social deficits and stereotyped, repetitive patterns of behaviors, limited interests, and cognitive impairment. Especially, social deficit has been considered a core feature of ASD. Because of the limitations of the experimental approach in humans, valid animal models are essential in an effort to identify novel therapeutics for social deficits in ASD. The genetic and environmental factors are clinically relevant to the pathophysiology of ASD...

Behavioral plasticity is subjected to various sensory stimuli, experiences, and physiological states, representing the temporal and spatial patterns of neural circuit dynamics. Elucidation of how genes and neural circuits in our brain actuate behavioral plasticity requires functional imaging during behavioral assays to manifest temporal and spatial neural regulation in behaviors. The exploration of the nervous systems of Caenorhabditis elegans has catalyzed substantial scientific advancements in elucidating the mechanistic link between circuit dynamics and behavioral plasticity...

This chapter aims to provide a comprehensive overview of the methodologies available to dissect genetic regulation of the nervous systems in the nematode Caenorhabditis elegans. These techniques encompass genetic screens and genetic tools to unravel the spatial-temporal contribution of genes on neural structure and function. Unbiased genetic screens on random mutations induced by ethyl methanesulfonate (EMS) or target gene silencing by genome-wide RNA interference (RNAi) help progress our understanding of the genetic control of neural development and functions...

Brain defects often lead to motor dysfunctions in humans. Drosophila melanogaster has been one of the most useful organisms in the study of neuronal biology due to its similarities with humans and has contributed to a more detailed understanding of the effects of genetic dysfunctions in the brain on behavior. We herein present modified protocols for the crawling assay with larvae and the climbing assay with adult flies that are simple to perform as well as a series of commands for ImageJ to automatically analyze data for the crawling assay...

Droplet digital PCR (ddPCR) is an emerging method for the absolute quantification of PCR products, and it can detect DNA copy numbers accurately. It analyzes the end-point absolute fluorescence signals of the PCR-positive droplets and calculates the target concentration. EvaGreen is a nonspecific double-stranded DNA-binding fluorescent dye, and the ddPCR system also supports assays using this cost-effective hydrolysis probe. Here, we describe a simple method of quantification for DNA copy numbers using the EvaGreen single-color fluorescent design...

Brain magnetic resonance imaging (MRI) is a noninvasive imaging modality that utilizes powerful magnets and radio waves to generate detailed images of the brain, making it a valuable tool for investigating malformations of cortical development (MCD). Various MRI techniques, including 3D T1-weighted, multiplanar thin-sliced T2-weighted, and 3D fluid-attenuated inversion recovery (FLAIR) sequences, can provide high-resolution images with excellent spatial and contrast resolution, allowing for a detailed visualization of cortical anatomy and abnormalities...

Malformations of cortical development (MCDs) are a diverse group of disorders that result from abnormal neuronal migration, proliferation, and differentiation during brain development. Head computed tomography (CT) has limited use in the diagnosis of MCDs and should be reserved for selected cases with specific indications or when magnetic resonance imaging is not available or contraindicated. CT can detect brain calcifications associated with MCDs, thus helping in the differential diagnosis between acquired and genetic MCDs or in the identification of different genetic patterns...

Many biological molecules in the brain interstitial fluid are involved in neuronal functions. Therefore, measuring the levels of these molecules in the extracellular fluid would provide deep insights into the physiological/pathological mechanisms underlying brain functions/disorders. In vivo microdialysis is a powerful technique used to examine the extracellular levels of various molecules in the brains of living animals. In neuroscience research, this technique has been widely used to investigate relatively small molecules including neurotransmitters and amino acids...

Measuring the membrane potential dynamics of neurons offers a comprehensive understanding of the molecular and cellular mechanisms that form their spiking activity, thus playing a crucial role in unraveling the mechanistic processes governing brain function. Techniques for intracellular recordings of membrane potentials pioneered in the 1940s have witnessed significant advancements since their inception. Among these, whole-cell patch-clamp recording has emerged as a leading method for measuring neuronal membrane potentials due to its high stability and broad applicability ranging from cultured cells to brain slices and even behaving animals...

The patch-clamp technique is one of the most useful tools to analyze the function of electrically active cells such as neurons. This technique allows for the analysis of proteins (ion channels and receptors), cells (neurons), and synapses that are the building blocks of neuronal networks. Cortical development involves coordinated changes in functional measures at each of these levels of analysis that reflect both cellular and circuit maturation. This chapter explains the technical and theoretical basis of patch-clamp methodology and introduces several examples of how this technique can be applied in the context of cortical development...