Chemical- and Viral-Based Transfection Methods

This section provides information on chemical transfection methods including liposome-meditated transfection, calcium phosphate, and viral mediated delivery.

Related Topics: Instrument-Based Transfection Methods, Posttransfection Analysis of Cells, and Cell Counting Methods.

Page Contents
 
Methods of Transfection
Method Function Recommended Cells Products
Lipid-mediated Uses lipids to cause a cell to absorb nucleic acids; transfer genetic material into the cell via liposomes, which are vesicles that can merge with the cell membrane Immortal cells, adherent (attached), or suspension cells TransFectin™ Lipid Reagent

SiLentFect™ Lipid Reagent for RNAi
Viral vector (for example, retrovirus, lentivirus, adenovirus, or adeno-associated viruses) Uses viral vectors to deliver nucleic acids into cells Attached adherent cells, stem cells, primary cells  

 

Advantages and Disadvantages of the Different Transfection Methods
  Advantages Disadvantages
Lipid Mediated
  • Efficiency — effectively deliver nucleic acids to cells in a culture dish
  • Minimal toxicity — deliver the nucleic acids with low cell death or little decrease in metabolism
  • Activity — transfected nucleic acids lead to measurable change
  • Easy to use — minimal steps required; adaptable to high-throughput systems
  • Economical — a more active lipid will reduce the cost of lipid and nucleic acid, and achieve effective results
  • Not applicable to all cell types — some cell lines are unable to transfect with lipids
Viral Mediated
  • Very high gene-delivery efficiency, 95–100%
  • Simplicity of infection
  • Labor intensive
  • Best for introducing a single cloned gene that is to be highly expressed
  • P2 containment required for most viruses
    • Institutional regulation and review boards required
    • Viral transfer of regulatory genes or oncogenes is inherently dangerous and should be carefully monitored
    • Host range specificity may not be adequate
  • Many viruses are lytic
  • Need for packaging cell lines
Calcium Phosphate
  • Inexpensive
  • High-efficiency cell type dependent
  • Can be applied to a wide range of cell types
  • Can be used for transient and stable transfection
  • Reagent consistency is critical for reproducibility
  • Small pH changes (±0.1) can compromise the efficacy
  • Size and quality of the precipitate are crucial to the success
  • of transfection
  • Calcium phosphate precipitation does not work in RPMI, due to the high concentration of phosphate within the medium
DEAE-Dextran
  • Inexpensive
  • Easy to perform and quick
  • Can be applied to a wide range of cell types
  • High concentrations of DEAE-dextran can be toxic to cells
  • Transfection efficiencies will vary with cell type
  • Can only be used with transient transfection
  • Typically produces less than 10% delivery in primary cells
Magnet Mediated
  • Rapid
  • Increased transfection efficiency by the directed transport, especially for low amounts of nucleic acids
  • High transfection rates for adherent mammalian cell lines and primary cell cultures (suspension cells and cells from other organisms also successfully transfected but need to be immortalized)
  • Mild treatment of cells
  • Can also be performed in the presence of serum
  • Relatively new method
  • Requires adherent cells; suspension cells need to be immobilized or centrifuged

The following table summarizes how common lipid and viral methods work.

Protocols for Different Transfection Methods
Lipid-Mediated
  • Cationic lipids are amphiphilic molecules that have a positively charged polar head group linked, via an anchor, to a nonpolar hydrophobic domain generally comprised of two alkyl chains
  • Structural variations in the hydrophobic domain of cationic lipids include the length and the degree of non-saturation of the alkyl chains
  • Electrostatic interactions between the positive charges of the cationic lipid head groups and the negatively charged phosphates of the DNA backbone are the main forces that allow DNA to spontaneously associate with cationic lipids
Viral Mediated
RNA Viruses
  • Retroviruses — a class of viruses that can create double-stranded DNA copies of their RNA genomes; these copies can be integrated into the chromosomes of host cells. Examples include:
    • Murine leukemia virus (MuLV)
    • Human immunodeficiency virus (HIV)
    • Human T-cell lymphotropic virus (HTLV)
DNA Viruses
  • Adenoviruses — a class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans; the virus that causes the common cold is an adenovirus
  • Adeno-associated viruses — a class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19
  • Herpes simplex viruses — a class of double-stranded DNA viruses that infect a particular cell type, neurons; herpes simplex virus type 1 is a common human pathogen that causes cold sores

Viral Transfection workflow.

Calcium Phosphate

The protocol involves mixing DNA with calcium chloride, adding the mixture in a controlled manner to a buffered saline/phosphate solution, and allowing the mixture to incubate at room temperature.

This step generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken up by the cells via endocytosis or phagocytosis.

Intercalation of Ca++ ions.

Protocol

Solution A: DNA in calcium solution
Solution B: 2x Hanks buffered saline solution

  • Add solution A to solution B while vortexing
  • Incubate 20–30 min. Apply the solution to the subconfluent cell culture
  • Incubate 2–12 hr. Replace the solution with complete growth medium
  • Assay for transient gene expression or begin selection for stable transformation

Cationic Polymers

Cationic polymers differ from cationic lipids in that they do not contain a hydrophobic moiety and are completely soluble in water. Given their polymeric nature, cationic polymers can be synthesized in different lengths, with different geometry (linear versus branched). The most striking difference between cationic lipids and cationic polymers is the ability of the cationic polymers to more efficiently condense DNA.

There are three general types of cationic polymers used in tranfections:

  • Linear (histone, spermine, and polylysine)
  • Branched
  • Spherical

Cationic polymers include polyethyleneimine (PEI) and dendrimers.


DEAE-Dextran

DEAE-dextran is a cationic polymer that tightly associates with negatively charged nucleic acids. The positively charged DNA:polymer complex comes into close association with the negatively charged cell membrane. DNA:polymer complex uptake into the cell is presumed to occur via endocytosis.

Protocol

Solution A: DNA (~1–5 µg/ml) diluted into 2 ml of growth medium with serum containing chloroquine
Solution B: DEAE-dextran solution (~50–500 ug/ml)
Solution C: ~5 ml of DMSO
Solution D: Complete growth medium

  • Add solution A to solution B, then mix gently
  • Aspirate cell medium and apply the mixed A and B solutions to the subconfluent cell culture. Incubate the DNA mixture for ~4 hr; check periodically for cell health
  • Aspirate the supernatant
  • Add solution C to induce DNA uptake
  • Remove DMSO and replace with solution D; assay for transient gene expression

Activated Dendrimers

Positively charged amino groups (termini) on the surface of the dendrimer molecule interact with the negatively charged phosphate groups of the DNA molecule to form a DNA-dendrimer complex.

The DNA-dendrimer complex has an overall positive net charge and can bind to negatively charged surface molecules on the membrane of eukaryotic cells. Complexes bound to the cell surface are taken into the cell by nonspecific endocytosis. Once inside the cell, the complexes are transported to the endosomes.

    • DNA is protected from degradation by endosomal nucleases by being highly condensed within the DNA-dendrimer complex.
    • Amino groups on the dendrimers that are unprotonated at neutral pH can become protonated in the acidic environment of the endosome. This leads to buffering of the endosome, which inhibits pH-dependent endosomal nucleases.

      Dendrimer molecule.

      Magnet-Mediated Transfection

      Magnet-mediated transfection uses magnetic force to deliver nucleic acids into target cells. Therefore, nucleic acids are first associated with magnetic nanoparticles. Then, application of magnetic force drives the nucleic acid-particle complexes towards and into the target cells, where the cargo is released.

      Magnet-mediated transfection.

       
      Transfection Protocols

      The transfection protocol online library contains protocols obtained from the literature, developed by Bio-Rad scientists, or submitted by scientists like you. Browse protocols to view our library and find your starting point or submit a protocol by clicking the proper technology.

      Documents MADHA

      Number Description Options
      5448
      MicroPulser Electroporator Flier, Rev A
      5582
      Gene Pulser Electroporation Buffer Product Information Sheet, Rev A
      5399
      Gene Pulser siRNA Electroporation References, Rev A
      5445
      Gene Pulser Xcell Electroporation System Flier, Rev A
      5598
      Gene Pulser MXcell Electroporation System Flier, Rev B
      5634
      Gene Pulser MXcell Electroporation System Brochure, Rev A
      1908
      Electroporation Cuvette Flier, Rev B
      5553
      Gene Modulation Workflow Brochure, Rev B
      1365
      Introducing Proteins Into Cells by Electroporation
      1345
      Electroporation of Primary Bone Marrow Cells
      1355
      Production of Hybridomas by Electrofusion
      1349
      Electroporation of T-Cell and Macrophage Cell Lines
      5542
      Electroporation Systems Brochure, Rev A
      [ Add to Cart (Free) ]
      5858
      The Gene Pulser MXcell Electroporation System Provides Reproducible Results in Electroporation Plates and Cuvettes With the Same Protocol, Rev A
      5641
      The Gene Pulser MXcell Electroporation System Delivers Consistent Results Required for Optimizing Delivery Protocols, Rev A
      5603
      Optimization of Electroporation Using Gene Pulser Electroporation Buffer and the Gene Pulser MXcell Electroporation System, Rev A
      5622
      Optimization of Electroporation Conditions With the Gene Pulser MXcell Electroporation System, Rev A
      5686
      Optimization of Electroporation Conditions for Jurkat Cells Using the Gene Pulser MXcell Electroporation System, Rev A
      5687
      Transfection of Mammalian Cells Using Preset Protocols on the Gene Pulser MXcell Electroporation System, Rev A
      5720
      Transfection of Neuroblastoma Cell Lines Using the Gene Pulser MXcell Electroporation System, Rev A
      5704
      Electroporation Conditions for Chinese Hamster Ovary Cells Using the Gene Pulser MXcell Electroporation System, Rev A
      5733
      Transfection of Chinese Hamster Ovary-Derived DG44 Cells Using the Gene Pulser MXcell Electroporation System, Rev A
      5774
      Delivery of siRNA by Electroporation Into Primary Human Neutrophils Using the Gene Pulser MXcell System, Rev A
      5778
      Electroporation Parameters for Transfection of HL-60 Leukocytic Cell Line With siRNA Using the Gene Pulser MXcell System, Rev B
      5823
      Electroporation of Primary Murine Mast Cells Using the Gene Pulser MXcell Electroporation System, Rev A
      5842
      Optimization of Electroporation Conditions for Two Different Burkitt Lymphoma Cell Lines Using the Gene Pulser MXcell System, Rev B
      5860
      Analysis of IL-4 Dependent Gene Expression in Namalwa Cells by siRNA Transfection: An Example of Pathway Analysis Using the Gene Pulser MXcell Electroporation System, Rev A
      5904
      Transfection of Mouse and Human Embryonic Stem Cells by Electroporation Using the Gene Pulser Mxcell™ System, Rev A
      5443
      Biolistic Particle Delivery Systems Brochure, Rev A
      5447
      Biolistic PDS-1000/He System Flier, Rev A
      5446
      Helios Gene Gun System Flier, Rev A
      2051
      Transformation of Filamentous Fungi by Microprojectile Bombardment
      2015
      Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cell Types
      2087
      Biolistic Transfection of Organotypic Brain Slices and Dissociated Cells
      2658
      Single-Cell Complementation of Barley mlo Mutants Using a PDS-1000/He Hepta System
      1688
      Optimization of Biolistic® Transformation Using the Helium-Driven PDS-1000/He System
      2433
      Transformation of Nematodes With the Helios Gene Gun
      2552
      The Gene Gun: Current Applications in Cutaneous Gene Therapy, Rev A
      2453
      Optimization of Gene Delivery Into Arabidopsis, Tobacco, and Birch Using the Helios Gene Gun System
      2531
      Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun
      2410
      Detection of Reporter Gene Activity in Cell Cultures and Murine Epidermis After Helios® Gene Gun-Mediated Particle Bombardment, Rev B
      2726
      Delivery of pCMV-S DNA Using the Helios® Gene Gun System Is Superior to Intramuscular Injection in Balb/c Mice
      1689
      Comparison of Performance Characteristics of Different Biolistic® Devices
      2768
      Biolistic Gene Transfer to Generate Transgenic Schistosomes, Rev A
      3105
      siLentFect Lipid Reagent Flier, Rev B
      2873
      TransFectin Lipid Reagent Brochure, Rev A
      2874
      TransFectin Lipid Reagent Flier, Rev B
      3197
      Optimization of TransFectin Lipid Reagent-Mediated Transfection for Different Cell Types, Rev A
      3138_015
      TransFectin Lipid Reagent Protocol, Human, A459, Lung Carcinoma
      3138_005
      TransFectin Lipid Reagent Protocol, Rat, PC12, Pheochromocytoma
      3138_009
      TransFectin Lipid Reagent Protocol, Human, 143B, Bone Marrow Osteosarcoma
      5226
      Highly Efficient Transfection of Mouse ES Cells With TransFectin Lipid Reagent
      3138_018
      TransFectin Lipid Reagent Protocol, Human, HEK 293, Kidney
      3138_017
      TransFectin Lipid Reagent Protocol, Human, HEK 293T, Kidney
      5439
      Highly Efficient Transfection of a Human Epithelial Cell Line With Chemically Synthesized siRNA Using siLentFECT Lipid Reagent, Rev A
      5370
      Transfection of Caco-2 Cells With siRNA Using the siLentFect Lipid Reagent, Rev A
      5808
      Novel Uses of Microarrays in Detecting Gene Silencing (Poster), Rev A

      TEST

      Number Description Options
      6179 Lipid Transfection Reagents Selection Guide
      Click to download