Genome editing is a powerful tool for establishing gene knockout or mutant cell lines. Here, we present a protocol for establishing knockout cell clones by deletion of large gene fragments using CRISPR-Cas9 with multiple guide RNAs. We describe steps for designing guide RNAs, cloning them into CRISPR-Cas9 vectors, cell seeding, transfection into cultured cells, clonal selection, and screening assays.
View Article and Find Full Text PDFEpithelial tissues cover the surfaces and lumens of the internal organs of multicellular animals and crucially contribute to internal environment homeostasis by delineating distinct compartments within the body. This vital role is known as epithelial barrier function. Epithelial cells are arranged like cobblestones and intricately bind together to form an epithelial sheet that upholds this barrier function.
View Article and Find Full Text PDFPodocytes serve as part of the renal filtration unit with slit diaphragms. Although the structure of slit diaphragms between two cells is well characterized, how the tricellular contact of podocytes is organized and how it changes in injured podocytes remains unknown. This study focused on a tricellular junction protein, angulin-3, and its localization in healthy podocytes, in developmental stages, and in pathologic conditions, using a newly established monoclonal antibody.
View Article and Find Full Text PDFTight junctions (TJs) are cell-cell junction structures critical for controlling paracellular permeability. On freeze-fracture replica electron microscopy, they appear as a continuous network of fibrils (TJ strands). TJ strands function as zippers that create a physical barrier against paracellular diffusion of molecules.
View Article and Find Full Text PDFTJs maintain the epithelial barrier by regulating paracellular permeability. Since TJs are under dynamically fluctuating intercellular tension, cells must continuously survey and repair any damage. However, the underlying mechanisms allowing cells to sense TJ damage and repair the barrier are not yet fully understood.
View Article and Find Full Text PDFOccludin, tricellulin, and marvelD3 belong to the tight junction (TJ)-associated MARVEL protein family. Occludin and tricellulin jointly contribute to TJ strand branching point formation and epithelial barrier maintenance. However, whether marvelD3 has the same function remains unclear.
View Article and Find Full Text PDFTight junctions (TJs) are composed of a claudin-based anastomosing network of TJ strands at which plasma membranes of adjacent epithelial cells are closely attached to regulate the paracellular permeability. Although the TJ proteins occludin and tricellulin have been known to be incorporated in the TJ strand network, their molecular functions remain unknown. Here, we established tricellulin/occludin-double knockout (dKO) MDCK II cells using a genome editing technique and evaluated the structure and barrier function of these cells.
View Article and Find Full Text PDFSince hepatitis C virus (HCV) is thought to enter into host hepatocytes using the same cellular pathways regardless of the genotypes, the host factors are promising targets to prevent and treat HCV infection. Human occludin (hOCLN) is one representative entry factor, and its second extracellular loop (EC2) contributes to the species selectivity of HCV-susceptibility. However, the exact function of hOCLN during HCV entry remains unknown, and no hOCLN-targeting antibodies or synthetic drugs that prevent and treat HCV infection have yet been developed.
View Article and Find Full Text PDFHere, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o) emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC) field to induce a linear cell-GUV alignment, and then a direct current (DC) pulse was applied to facilitate transient electrofusion.
View Article and Find Full Text PDFContact guidance is a cellular phenomenon observed during wound healing and developmental patterning, in which adherent cells align in the same direction due to physical cues. Despite numerous studies, the molecular mechanism underlying the consistent cell orientation is poorly understood. Here we fabricated microgrooves with a pitch of submicrons to study contact guidance of smooth muscle cells.
View Article and Find Full Text PDFThe micropatterning of cells, which restricts the adhesive regions on the substrate and thus controls cell geometry, is used to study mechanobiology-related cell functions. Plasma lithography is a means of providing such patterns and uses a spatially-selective plasma treatment. Conventional plasma lithography employs a positionally-fixed mask with which the geometry of the patterns is determined and thus is not suited for producing on-demand geometries of patterns.
View Article and Find Full Text PDFSheets of cells can be used for tissue regenerative medicine. Cell alignment within the sheet is now a key factor in the next generation of this technology. Anisotropic cell sheets without random cell orientations have been conventionally produced with photolithographically, microfabricated substrates using special facilities and equipment.
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