Researchers have a wide variety of gene delivery techniques to introduce plasmid DNA, siRNA or duplex RNAi, oligonucleotides, and RNA into eukaryotic cells for a variety of research and drug discovery applications. A review of these techniques is provided on the table below

Lipid delivery using cationic lipids

How it works:

DNA, siRNA, or oligonucleotides and transfection reagent
(commercially available) are diluted in separate tubes.



DNA and transfection reagent combined to form complexes.
Positive charge on cationic lipid binds to phosphate backbone on nucleic acid.



Complexes added to cells. Positive charge on cationic lipid helps bind complex to membrane.



Complexes enter the cell via endocytosis.



Assay for gene expression or silencing.

Advantages

  • Commercially available with reproducible results
  • Applicable to a broad range of cell lines
  • High efficiency and expression performance
  • Fast and easy protocols which do not require media changes

Disadvantages

  • Optimization may be necessary—some cell lines are sensitive to cationic lipids
  • Some cell lines are not readily transfected with cationic lipids

Viral delivery

How it works:

Generate recombinant virus via gene cloning, cell culture, virus isolation,
and clone characterization.



Prepare and purify high titer viral vectors.



Infect cells (containing viral receptor) at appropriate multiplicity of infection (MOI).



Remove virus and/or add fresh medium.



Assay for gene expression or silencing.

Advantages

  • Works well for difficult-to-transfect cell types

Disadvantages

  • Technically challenging and time consuming to generate recombinant viruses
  • Cell lines to transfect must contain viral receptors
  • Safety considerations

Delivery by electroporation

How it works:

Prepare cells by suspending in electroporation buffer.
Pulse cells electrically in the presence of specialized buffer and DNA.



Electrical pulse creates a potential difference across the membrane and induces
temporary pores in the cell membrane for DNA entry



Cells returned to growth conditions.



Assay for gene expression or silencing.

Advantages

  • Can work well for difficult-to-transfect cell types

Disadvantages

  • Requires instrument
  • Optimization of electrical pulse and field strength parameters required
  • Significantly more manipulation of cells required
  • High toxicity levels may be observed
  • Can irreversibly damage the membrane and lyse the cells

Delivery by other chemical methods, i. e. calcium phosphate precipitation, DEAE-dextran, polybrene

How it works:

Prepare solutions. Mix calcium chloride (in phosphate buffer) and DNA.



Allow precipitation of extremely small, insoluble particles containing condensed DNA



CaPO 4: Add complexes to cell culture and incubate.
Complexes adhere to cell membranes, and enter into the cytoplasm by endocytosis.



DEAE-Dextran and polybrene:
Complexes are delivered by osmotic shock using DMSO or glycerol. 
Positive-charged polymers complex with negatively charged DNA molecules to enable binding to the cell surface.



Remove complexes from cells, wash, and add fresh culture medium.



Assay for gene expression or silencing.

Advantages

  • Reagents costs are inexpensive

Disadvantages

  • Requires careful preparation of reagents—CaPO4 solutions are pH sensitive
  • DEAE-dextran is limited to transient transfections
  • Cell toxicity
  • Reproducibility can be problematic

For Research Use Only. Not for use in diagnostic procedures.