Transfection & Gene Editing

Transfected cells for protein expression and gene function studies

Transfection is the process of introducing nucleic acids into eukaryotic cells. Cells can be stably transfected for the integration of DNA into their genome, or transiently transfected for protein expression of temporary duration. Chemical, physical, and biological methods are used to transfect cells, enabling the study of gene function and expression within a cellular environment. Applications include gene therapy, generation of induced pluripotent stem cells (iPSC), gene silencing by RNA interference (RNAi), and the production of therapeutic antibodies and proteins.

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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 destabilize 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.

  • 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.