Developmental Biology


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0 Q&A 991 Views Jan 20, 2025

Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult. Previously described methods for MEF immortalization are often inconsistent or alter the physiological properties of the cells. Here, we describe an optimized method that overcomes these limitations. By using electroporation to deliver CRISPR constructs that target the Tp53 gene, the method reliably generates immortalized MEFs (iMEFs) within three weeks. Importantly, iMEFs closely resemble the parent cell populations, and individual iMEFs can be cloned and expanded for subsequent genetic manipulation and characterization. We envision that this protocol can be adopted broadly to immortalize other mouse primary cell types.

0 Q&A 369 Views Jan 20, 2025

The fate mapping technique is essential for understanding how cells differentiate and organize into complex structures. Various methods are used in fate mapping, including dye injections, genetic labeling (e.g., Cre-lox recombination systems), and molecular markers to label cells and track their progeny. One such method, the FlashTag system, was originally developed to label neural progenitors. This technique involves injecting carboxyfluorescein diacetate succinimidyl ester (CFSE) into the lateral ventricles of mouse embryos, relying on the direct uptake of dye by cells. The injection of CFSE into the lateral ventricle allows for the pulse labeling of mitotic (M-phase) neural progenitors in the ventricular zone and their progeny throughout the brain. This approach enables us to trace the future locations and differentiation paths of neural progenitors. In our previous study, we adapted this method to selectively label central nervous system–associated macrophages (CAMs) in the lateral ventricle by using a lower concentration of CFSE compared to the original protocol. Microglia, the brain's immune cells, which play pivotal roles in both physiological and pathological contexts, begin colonizing the brain around embryonic day (E) 9.5 in mice, with their population expanding as development progresses. The modified FlashTag technique allowed us to trace the fate of intraventricular CAMs, revealing that certain populations of microglia are derived from these cells. The optimized approach offers deeper insights into the developmental trajectories of microglia. This protocol outlines the modified FlashTag method for labeling intraventricular CAMs, detailing the CFSE injection procedure, evaluation of CFSE dilution, and preparation of tissue for immunohistochemistry.

0 Q&A 310 Views Dec 20, 2024

In this paper, we present a detailed protocol for microinjecting DNA, RNA, or protein solutions into fertilized eggs of the multicolored Asian ladybird beetle, Harmonia axyridis, under a stereomicroscope equipped with an injection apparatus. H. axyridis is an emerging model organism for studying various biological fields, showing intraspecific polymorphisms exhibiting highly diverse color patterns on the elytra. Here, we describe how to rear ladybird beetles in a laboratory and obtain fertilized eggs for microinjection experiments. We also provide a constant fluid flow injection method, which enhances the efficiency of microinjection and improves throughput. Our step-by-step protocol is applicable to generating transgenic or genome-edited ladybird beetles, facilitating functional genetics in H. axyridis; the microinjection method should be applicable to other insect eggs.

0 Q&A 519 Views Dec 20, 2024

Zebrafish and medaka are valuable model vertebrates for genetic studies. The advent of CRISPR-Cas9 technology has greatly enhanced our capability to produce specific gene mutants in zebrafish and medaka. Analyzing the phenotypes of these mutants is essential for elucidating gene function, though such analyses often yield unexpected results. Consequently, providing researchers with accessible and cost-effective phenotype analysis methods is crucial. A prevalent technique for investigating calcified bone development in these species involves using transgenic fish that express fluorescent proteins labeling calcified bones; however, acquiring these fish and isolating appropriate crosses can be time-consuming. We present a comprehensive protocol for visualizing ossified bones in zebrafish and medaka larvae and juveniles using calcein and alizarin red S staining, which is both economical and efficient. This method, applicable to live specimens during the ossification of bones, avoids apparent alterations in skeletal morphology and allows for the use of different fluorescent dyes in conjunction with transgenic labeling, thus enhancing the analysis of developmental processes in calcifying bones, such as vertebrae and fin rays.

0 Q&A 252 Views Dec 5, 2024

Developing a physiologically relevant in vitro model of the respiratory epithelium is critical for understanding lung development and respiratory diseases. Here, we describe a detailed protocol in which the fetal mouse proximal epithelial progenitors were differentiated into 3D airway organoids, which contain terminal-differentiated ciliated cells and basal stem cells. These differentiated airway organoids could constitute an excellent experimental model to elucidate the molecular mechanisms of airway development and epithelial cell fate determination and offer an important tool for establishing pulmonary dysplasia disease in vitro.

0 Q&A 277 Views Nov 5, 2024

Maternal mRNAs and proteins are produced during oogenesis by more than 60% of zebrafish genes. They are indispensable for fertilization and early embryogenesis. Generation and analysis of the maternal mutant is the most direct way to characterize the maternal function of the specific gene. However, due to the lethality of zygotic mutants, the maternal function of most genes in zebrafish remains elusive. Several methods have been developed to circumvent this obstacle, including mRNA rescue, germ-line replacement, oocyte microinjection in situ, mosaic mutation, and bacterial artificial chromosome (BAC)-mediated conditional rescue. Here, we provide an alternative approach to generate zebrafish maternal mutants rapidly and efficiently by introducing four tandem sgRNA expression cassettes into Tg(zpc:zcas9) embryos. This method is more technically feasible and cost- and time-effective than other established methods.

0 Q&A 403 Views Nov 5, 2024

Osteoclasts are terminally differentiated multinucleated giant cells that mediate bone resorption and regulate skeletal homeostasis under physiological and pathological states. Excessive osteoclast activity will give rise to enhanced bone resorption, being responsible for a wide range of metabolic skeletal diseases, ranging from osteoporosis and rheumatoid arthritis to tumor-induced osteolysis. Therefore, the construction of in vitro models of osteoclast-mediated bone resorption is helpful to better understand the functional status of osteoclasts under (patho)physiological conditions. Notably, it is essential to provide an in vivo–relevant bone substrate that induces osteoclasts to generate authentic resorption lacunae and excavate bone. Here, we summarize the experimental design of a reproducible and cost-effective method, which is suitable for evaluating the regulatory mechanisms and influence of molecular agonists and antagonists as well as therapeutics on osteoclast-mediated bone-resorbing activity.

0 Q&A 277 Views Oct 20, 2024

Neuroscience incorporates manipulating neuronal circuitry to enhance the understanding of intricate brain functions. An effective strategy to attain this objective entails utilizing viral vectors to induce varied gene expression by delivering transgenes into brain cells. Here, we combine the use of transgenic mice, neonatal transduction with adeno-associated viral constructs harboring inhibitory designer receptor exclusively activated by designer drug (DREADD) gene, and the DREADD agonist clozapine N-oxide (CNO). In this way, a chemogenetic approach is employed to suppress neuronal activity in the region of interest during a critical developmental window, with subsequent investigation into its effects on the neuronal circuitry in adulthood.

0 Q&A 284 Views Oct 20, 2024

The mammary gland undergoes functional, developmental, and structural changes that are essential for lactation and reproductive processes. An overview of such unique tissue can offer clearer insights into mammary development and tumorigenesis. Compared to traditional methods, mouse mammary gland whole mount is a pivotal technique that provides three-dimensional structural perspectives on gland morphology and developmental stages, offering an inexpensive and accessible approach. This protocol outlines the tissue isolation of the mouse mammary gland and provides detailed instructions for whole-mount staining and analysis. Mammary gland tissues are carefully dissected from euthanized mice and stained with Carmine Alum to highlight the ductal structures, enabling detailed visualization of the branching patterns and morphological changes. Light microscopy is used to capture a panoramic image of the stained mammary gland, enabling the quantitative analysis of terminal end buds (TEBs) and bifurcated TEBs to further investigate mammary gland remodeling. This method can provide invaluable insights, particularly in the study of mammary gland morphogenesis and tumorigenesis, underscoring its significance in both basic research and clinical applications.

0 Q&A 445 Views Oct 20, 2024

Enzyme-catalyzed proximity labeling is a potent technique for the discernment of subtle molecular interactions and subcellular localization, furnishing contextual insights into the protein of interest within cells. Although ascorbate peroxidase2 (APEX2) has proven effective in this approach when overexpressed, its compatibility with endogenous proteins remains untested. We improved this technique for studying native protein–protein interactions in live Drosophila ovary tissue. Through CRISPR/Cas9 genome editing, APEX2 was fused with the endogenous dysfusion gene. By pre-treating the tissue with Triton X-100 to enhance biotin-phenol penetration, we successfully identified multiple proteins interacting with the native Dysfusion proteins that reside on the inner nuclear membrane. Our protocol offers a comprehensive workflow for delineating the interactome networks of ovarian components in Drosophila, aiding future studies on endogenous protein–protein interactions in various tissues of other animals.




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