The cell membrane consists of a phospholipid bilayer with embedded proteins and carries a net negative charge. Thus, it presents an impenetrable barrier to large molecules that, like the phosphate backbones of DNA and RNA, are also negatively charged. To sneak nucleic acids through the cell membrane, researchers have developed a number of techniques each using a different approach—from using chemicals and carrier molecules that coat the nucleic acids to neutralize them to physical methods that create transient pores in the membrane to introduce the DNA directly into the cell.

Gene delivery technologies

Transfection technologies available today can be broadly classified into three groups: chemical, biological, and physical. No one method can be applied to all cells and all experiments. The ideal approach should be selected depending your cell type and experimental needs, should have high transfection efficiency, low cell toxicity, and minimal effects on normal physiology, and be easy to use and reproducible (Kim and Eberwine, 2010).

Chemical

Chemical methods that use carrier molecules to neutralize or impart a positive charge to the negatively charged nucleic acids and include:

Biological

Biological methods that rely on genetically engineered viruses to transfer non-viral genes into cells (also known as transduction) and include:

Physical

Physical methods directly deliver nucleic acids into the cytoplasm or the nucleus of the cell and include:

Compare chemical gene delivery methods
Technology Advantages Disadvantages
Cationic lipid mediated delivery
  • Fast and easy protocols
  • Commercially available with reproducible results
  • High efficiency and expression performance
  • Applicable to a broad range of cell lines and high-throughput screens
  • Can be used for delivering DNA, RNA, and protein
  • No size limitation on the packaged nucleic acid
  • Applicable to both transient and stable protein production
  • Can be used for in vivo delivery of nucleic acids
  • Optimization may be necessary—some cell lines are sensitive to cationic lipids
  • Some cell lines are not readily transfected with cationic lipids
  • Presence of serum may interefere with complex formation and lower transfection efficiency
  • Absence of serum in the medium may increase cytotoxicity
Calcium phosphate co-precipitation
  • Inexpensive and easily available
  • Applicable to both transient and stable protein production
  • High efficiency (cell line dependent)
  • Requires careful preparation of reagents—CaPO4 solutions are sensitive to changes in pH, temperature, and buffer salt concentrations
  • Reproducibility can be problematic
  • Cytoxicity, especially in primary cells
  • Does not work with RPMI due to high phosphate concentration of the medium
  • Not suited for in vivogene transfer to whole animals
DEAE-dextran
  • Relatively simple technique
  • Reproducible results, inexpensive
  • Chemical cytoxicity in some cell types
  • Limited to transient transfection
  • Low transfection efficiency
Delivery by other cationic polymers (e.g., polybrene, PEI, dendrimers)
  • Typically stable in serum and not temperature sensitive
  • High efficiency (cell line dependent), reproducible results
  • Cytoxicity in some cell types
  • Non-biodegradable (dendrimers)
  • Limited to transient transfection
Compare biological gene delivery methods
Technology Advantages Disadvantages
Viral delivery
  • Highest efficiency amongst gene delivery methods (80–90% transduction efficiency in primary cells)
  • Works well with difficult to transfect cell types
  • Can be used for in vivo delivery of nucleic acids
  • Can be used for making stable cell lines (retroviral vectors) or for transient expresssion (adenoviral vectors)
  • Cell lines to transfect must contain viral receptors
  • Limited insert size (~10 kb for most viral vectors versus ~100 kb for non-viral vectors)
  • Technically challenging and time consuming to generate recombinant viruses
  • Present biosafety issues (activation of latent disease, immunogenic reactions, cytotoxicity, insertional mutagenesis, malignant transformation of cells)
Compare physical gene delivery methods
Technology Advantages Disadvantages
Electroporation
  • Simple principle
  • Reproducible results after optimization
  • No need for vector
  • Less dependent on cell type and condition
  • Rapid transfection of large number of cells after optimization
  • Requires special instrument
  • Optimization of electrical pulse and field strength parameters required
  • Significantly more manipulation of cells required
  • High toxicity levels may be observed
  • High mortality rate requires large number of cells
  • Can irreversibly damage the membrane and lyse the cells
Biolistic particle
delivery
(particle
bombardment)
  • Less dependent on cell type and condition
  • Can be used for in vivo delivery of nucleic acids
  • Straightforward method with reliable results
  • No limitation to the size and or number of genes that can be delivered
  • Primarily used for genetic vaccination and agricultural
    application
  • Requires expensive instrument
  • Causes physical damage to samples
  • High mortality rate requires large number of cells
  • Preparation of microparticles is required
  • Relatively costly for research applications
  • Generally less efficient than electroporation or viral- or lipid-mediated delivery
Direct
microinjection
  • Less dependent on cell type and condition
  • Allows single-cell transfection
  • Straightforward method with reliable results
  • No limitation to the size and or number of genes that can be delivered
  • No need for vector
  • Requires expensive instrument
  • Technically demanding and very labor-intensive (one cell at a time)
  • Often causes cells deaths
Laser-mediated
transfection

(phototransfection)
  • Can be used for delivering DNA, RNA, proteins, ions, dextrans, small molecules, and semiconductor
    nanocrystals
  • Can be applied to very small cells
    Allows single-cell transfection or transfection of large number of cells at the same time
  • No need for vector
  • High efficiency
  • Applicable to a broad range of cell lines
  • Requires expensive laser-microscope system
  • Requires cells to be attached
  • Technically demanding