Name: ビデオ: 接着培養細胞に核酸を導入するためのビーズローディング:接着性哺乳動物細胞に蛍光タンパク質をコードするプラスミドをロードする技術 - 概念
Uploaded: 2023-04-30T14:56:35.000+00:00
Duration: 1 min 53 s
Description: 1.6K ビュー. 出典:Cialek, C. A. et al.接着性ヒト細胞へのタンパク質および核酸のローディングをビーズで行います。J. Vis. Exp.(2021年)。 この動画では、ビーズローディングを使用して付着した哺乳類細胞に核酸を導入する手順を説明します。

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1. Bead loading cells
NOTE: If required, wash the cells briefly with phosphate-buffered saline (PBS) and then add 2 mL of the optimal medium. Incubate for at least 30 min.
<ol>
	<li>Make a solution of 3-8 &#181;L containing the desired plasmids, protein, and/or particles. Use ~1 &#181;g (0.1-1 pmol) of each type of plasmid and ~0.5 &#181;g (0.01 nmol) of protein, depending on experimental requirements. Use a low-retention tube for proteins so that they are not left behind on the tube walls. Bring the solution up to a minimum of 3 &#181;L with PBS, and adjust the solution volume to coat the entire area of cells to be loaded (i.e., the chamber&#39;s microwell, Figure 1B).</li>
	<li>Mix the solution thoroughly by pipetting up and down and/or flicking the tube. Briefly spin the solution down to the bottom of the tube in a tabletop microfuge.</li>
	<li>Transfer the bead loading solution and the chamber of cells into a tissue culture hood. Perform the remaining steps in the tissue culture hood using sterile technique.</li>
	<li>Remove the medium from the cells and temporarily store it in a sterile tube. Gently aspirate all medium from around the edges of the chamber and tilt the chamber at approximately a 45&#176; angle and remove the remaining drop of media in the center microwell. During medium removal, make sure to avoid letting the pipette tip touch the glass, which may result in cell peeling and loss. Move quickly to the next step so that the cells are not dry for long.</li>
	<li>Gently pipette the bead loading solution onto the glass microwell in the center of the chamber. Optional: Incubate with gentle rocking for ~30 s without allowing the chamber to dry up completely.</li>
	<li>Gently disperse a monolayer of glass beads on top of the cells, preferably using a bead loading apparatus (Figure 1A). Ensure that the beads cover the cells in the glass-bottom microwell completely.</li>
	<li>Pinching the chamber with two fingers, tap it against the hood surface by lifting it ~2 inches and bringing it down firmly. Use a force approximately equivalent to dropping the dish from that height. Repeat for a total of ~10 taps. 
	NOTE: Ensure that the taps do not substantially peel the cells. Tapping can be optimized for the cell type. If cells load poorly, tap harder; however, if many cells peel off, tap more lightly.</li>
	<li>Gently add medium back into the chamber by pipetting slowly onto the plastic side of the chamber. Try to aspirate any floating beads without disturbing the cells. Add more pre-warmed media at this step if too much was removed. Incubate the cells for 0.5-2 h in the incubator.</li>
</ol>

<table><tbody><tr><td>10 cm cell culture dishes</td><td>VWR</td><td>82050-916</td><td>Use to culture cells</td></tr><tr><td>35 mm cell culture dishes</td><td>Falcon</td><td>353001</td><td>Use to construct bead loader</td></tr><tr><td>Attofluor Cell Chamber</td><td>Thermo Fisher Scientific</td><td>A7816</td><td>Use to construct the custom bead loader</td></tr><tr><td>DMEM, high glucose, no glutamine</td><td>Thermo Fisher Scientific</td><td>11960069</td><td>Use in general cell culture</td></tr><tr><td>Glass bottom dishes, 35 mm, #1.5, 14 mm glass</td><td>MatTek Corporation</td><td>P35G-1.5-14-C</td><td>Seed cells onto these chambers for imaging</td></tr><tr><td>Glass beads, acid washed, &amp;le;106 &amp;micro;m</td><td>Millipore Sigma</td><td>G4649</td><td>Sprinkle on cells to bead load plasmid DNA and proteins</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Thermo Fisher Scientific</td><td>AM9625</td><td>Working stock of sterile 1X PBS</td></tr><tr><td>Phenol-free DMEM</td><td>Thermo Fisher Scientific</td><td>31053036</td><td>Use on cells before imaging</td></tr><tr><td>Opti-MEM, Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td>Optimal media for incubating cells before bead loading (optional step)</td></tr></tbody></table>

bead loading to introduce nucleic acids into adherent cultured cells: a technique to load fluorescent protein-encoding plasmids into adherent mammalian cells

Source: Cialek, C. A. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/62559">Bead Loading Proteins and Nucleic Acids into Adherent Human Cells.</a> J. Vis. Exp. (2021).
In this video, we describe the procedure to introduce nucleic acids into adherent mammalian cells using bead loading.

<img alt="Figure 1" src="/files/ftp_upload/20985/20985_Figure1.jpg" /> 
Figure 1: Bead loading apparatus, technique, and timeline

Bead Loading to Introduce Nucleic Acids Into Adherent Cultured Cells: A Technique to Load Fluorescent Protein-Encoding Plasmids Into Adherent Mammalian Cells

Source: Cialek, C. A. et al. Bead Loading Proteins and Nucleic Acids into Adherent Human Cells. J. Vis. Exp. (2021).
In this video, we describe the ...

Begin with a culture of adherent mammalian cells in a culture plate. Transfer sterilized alkali-treated, micron-sized glass beads into the chamber of a customized bead loader apparatus. Cover the chamber opening using a mesh with optimum-size openings to allow the beads to pass through during loading. Secure the mesh with an imaging chamber assembly.
Remove media from the plate. Pipette a suspension of fluorescent reporter protein-encoding plasmid DNA into the plate for even distribution over the cells. Using the bead loader apparatus, gently disperse a monolayer of glass beads onto the cells. Tap the culture plate briefly with suitable force.
The glass beads briefly roll on top of the adherent cells, while the alkali treatment makes them less adherent to the cells. The impact of collisions between beads and cells transmits adequate strain creating localized disruptions in the cellular membranes. These formed transient pores of small dimensions facilitate the uptake of plasmid DNA into the cells while preventing the entry of large beads.
During the recovery period, the cellular membrane reseals, restoring the membrane integrity and entrapping the plasmid DNA. Add media to the plate and carefully aspirate any visible floating beads from the plate. Incubate the plate.
Successfully transfected cells containing plasmid DNA express the fluorescent reporter proteins, which can be visualized under a fluorescence microscope.

bead-loading-to-introduce-nucleic-acids-into-adherent-cultured-cells

Universidad de Cartagena UDEC

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JoVE Encyclopedia of Experiments

Biological Techniques

Gene Transfer Techniques

Transfection

1.6K ビュー. 出典:Cialek, C. A. et al.接着性ヒト細胞へのタンパク質および核酸のローディングをビーズで行います。J. Vis. Exp.(2021年)。 この動画では、ビーズローディングを使用して付着した哺乳類細胞に核酸を導入する手順を説明します。 

ビデオ: 接着培養細胞に核酸を導入するためのビーズローディング:接着性哺乳動物細胞に蛍光タンパク質をコードするプラスミドをロードする技術 - 概念

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

JoVE Journal

発生生物学

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

細菌接合に関与する転写タンパク質の配列特異的分析のための接合交配アッセイ

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

遺伝学

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

Bead Loading to Introduce Nucleic Acids Into Adherent Cultured Cells: A Technique to Load Fluorescent Protein-Encoding Plasmids Into Adherent Mammalian Cells

記録

Bead Loading to Introduce Nucleic Acids Into Adherent Cultured Cells: A Technique to Load Fluorescent Protein-Encoding Plasmids Into Adherent Mammalian Cells