Transfection & Gene Editing
Transfection is the process of introducing nucleic acids, such as DNA or RNA, into eukaryotic cells, allowing for the manipulation of gene expression within a cellular environment. This can be achieved through stable transfection, where the DNA is integrated into the cell's genome, or transient transfection, where protein expression occurs for a limited time. Various methods, including chemical, physical, and biological techniques, are employed to transfect cells, enabling researchers to study gene function and expression. Transfection has a broad range of applications, including gene therapy, the generation of induced pluripotent stem cells (iPSCs), gene silencing through RNA interference (RNAi), and the production of therapeutic antibodies and proteins. These applications make transfection a crucial tool in both basic research and clinical development.
Overview
Common Transfection Methods
- Lipids and liposomes: Cationic lipids form liposomes containing DNA or RNA for delivery. These liposomes fuse with the cell membrane and release nucleic acid into the cell.
- Calcium phosphate: Calcium phosphate facilitates the binding of DNA to the cell surface, allowing genetic material to enter the cell by endocytosis.
- Cationic polymers: In polymer-based transfection, exogenous DNA forms complexes with cationic polymers such as polyethylenimine (PEI) that enter host cells by endocytosis.
- Lentiviral transduction: Cells are infected with modified lentivirus vectors, which convert their viral RNA to double stranded DNA for integration into the host genome for delivery.
- Microinjection: Target cells are first positioned under a microscope. Nucleic acid is then directly injected into the cytoplasm or nucleus using a fine glass capillary needle.
- Electroporation: Cells are exposed to high-intensity electric current that destabilizes membranes, increasing their permeability for gene delivery.
Transfection is routinely used in gene editing and gene silencing techniques that have enhanced our understanding of complex biological processes and enabled the use of gene therapy to treat disease.
Gene Editing Techniques
CRISPR-Cas systems exploit a bacterial defense mechanism that uses genetic CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) coupled with Cas (CRISPR-associated) endonucleases to cut genomic DNA at targeted positions and remove or replace genes in vivo.
Engineered zinc finger nucleases (ZFNs) are constructed of DNA binding domains and endonucleases and cleave DNA at targeted sites for gene editing.
RNAi reagents such as short hairpin RNAs (shRNAs) and small interfering RNAs (siRNAs) limit gene transcript levels for gene silencing by either suppressing transcription or activating sequence-specific RNA degradation processes.
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