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Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins
Published on: November 30, 2021
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Single cell/cellular subregion-targeted phototransfection.
Jaehee Lee1, Jai-Yoon Sul1, James H Eberwine2
1Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Cold Spring Harbor Protocols
|September 4, 2014
Summary
Phototransfection uses a laser to create temporary cell membrane pores for precise genetic material delivery. This method enables targeted nucleic acid introduction into specific cells or cell regions, overcoming limitations of traditional techniques.
Area of Science:
- Cell biology
- Molecular biology
- Biotechnology
Background:
- Introducing exogenous genetic material (DNA, mRNA, siRNA, miRNA) into cells is crucial for studying biological functions.
- Standard methods like lipid-mediated transfection and microinjection have limitations in specificity and labor intensity, especially for polarized cells like neurons.
Purpose of the Study:
- To introduce a novel method, phototransfection, for precise and efficient delivery of exogenous genetic material into cells.
- To overcome the limitations of existing transfection methods regarding regional specificity and cell type versatility.
Main Methods:
- Phototransfection utilizes a laser-microscope system to induce transient pores in the plasma membrane of target cells.
- The induced pores allow diffusion of genetic material (e.g., mRNA) into the cytosol before resealing.
- The technique is adaptable for various cell types and genetic materials, including simultaneous transfection of multiple mRNAs.
Main Results:
- Phototransfection allows for targeted delivery of genetic material to specific cells or subregions within a cell with minimal damage.
- The method demonstrated successful transfection of green fluorescent protein (GFP) mRNA into a single neuronal process.
- Optimization guidelines and key steps for performing phototransfection are provided.
Conclusions:
- Phototransfection offers a versatile, regionally specific, and efficient alternative to conventional transfection methods.
- This technique enhances the study of gene function, particularly in challenging cell types like neurons.
- The protocol facilitates precise control over the amount and location of nucleic acid delivery.
