Introduction to Transfection

* This product is for research use only. Not intended for use in the treatment or diagnosis of disease.

What is transfection?

Broadly speaking, transfection is the process of introducing nucleic acid (DNA or RNA) into eukaryotic cells by non-viral methods. This introduction of foreign nucleic acids using various chemical, biological, or physical methods can lead to changes in cell properties, thereby allowing the study of gene function and protein expression in the context of cells.

Transfection overcomes the inherent challenges of introducing negatively charged molecules (for example, the phosphate backbone of DNA and RNA) into cells with negatively charged membranes. In transfection, the introduced nucleic acid may temporarily exist in the cell so that it can only be expressed for a limited time without replication, or it can be stablilized and integrated into the genome of the recipient and replicated in the host genome.

Transfection Terminology

TransfectionTransformationTransduction
Definition Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. However, its present meaning includes any artificial introduction of foreign nucleic acid into a cell.Transformation is often used to describe the non-viral DNA transfer in bacteria, non‑animal eukaryotic cells, and plant cells.Transduction is used to describe virus-mediated DNA transfer.

Advantages and Disadvantages of the Different Transfection Methods

Methods  Advantages Disadvantages Recommended cellsBOC Sciences Solutions
Chemical
Lipid mediated
  • High efficiency-able to effectively deliver nucleic acids into cells and obtain measurable changes 
  • Low cytotoxicity
  • Easy to use-minimum steps required; adapt to high throughput systems
  • Economical-more active lipids can reduce costs and obtain effective results
  • Not suitable for all cell types-some cell lines cannot be transfected by lipofection
Immortal cells, adherent (attached) or suspended cellsMost of our products are based on cationic lipid
Calcium phosphate
  • High-efficiency
  • Cheap
  • Can be applied to multiple cell types
  • Small pH changes (±0.1) may affect the efficiency
  • The size and quality of the sediment are critical to success
Adherent and nonadherent cell lines.Calcium Phosphate Transfection Reagent
Cationic Polymers (e.g., PEI)
  • No viral vector
  • Chemical toxicity to some cell types
Tumor cell linesPEI Transfection Reagent
DEAE-Dextran
  • Cheap
  • Easy to perform and quick
  • Suitable to a wide range of cell types
  • High concentrations of DEAE-dextran may be toxic to cells
  • Different transfection efficiency in different cell types
  • Can only be used for transient transfection
  • Poor performance in primary cells (usually < 10%)
Magnet mediated
  • Fast
  • Improve transfection efficiency through targeted transport, especially for small amounts of nucleic acids
  • Gentle treatment of cells
  • Relatively new method
  • Need adherent cells; suspension cells need to be fixed or centrifuged
Immortal cells, Adherent mammalian cell lines and primary cells
Biological
Viral mediated
  • Very high gene delivery efficiency, 95-100%
  • Easy to use
  • Effective for dissociated cells, slices, and in vivo
  • Most viruses need P2 containment
  • Risk of foreign gene integration into the host genome
  • Many viruses are lytic
  • Requires packaging cell line Immunogenicity
  • DNA package size limit
Attached adherent cells, stem cells, primary cells
Physical
Electroporation
  • No need for vector
  • Easy and rapid
  • Not alter target cell morphology and functions
  • Demands experimenter skill, laborious procedure
  • Need to determine the optimum electroporation conditions
  • Can cause cell death if transfection is not performed under optimum conditions
A variety of cells (especially cells that are difficult to transfect, such as primary and stem cells)Electroporation Products

Factors affecting transfection efficiency

  • Cell passage number
  • Keep the number of passages<50. In addition, the number of cell passages used in various experiments should be consistent.

  • Cell state
  • Cells should be grown in a medium suitable for the cell line and supplemented with serum or growth factors according to survival needs. Contaminated cells and media must not be used for transfection. If the cells are damaged in any way, they should be discarded and re-seeded from frozen, uncontaminated stock.

  • Cell density
  • Normally, the confluency of the transfected cells is 40-80%. Too few cells will cause the culture to grow poorly without cell-to-cell contact. Too many cells can lead to contact inhibition, which prevents the cells from absorbing foreign DNA.

  • Nucleic Acid Quality and Quantity
  • The plasmid DNA used for transfection should be free of protein, RNA, chemical, and microbial contamination, and the appropriate final concentration is 0.2-1mg/ml. The optimal amount of DNA used for transfection depends on the type of DNA, transfection reagent, target cell line, and the number of cells.

  • Cytotoxicity of transfection reagent
  • When choosing a transfection method or reagent, both the transfection efficiency and the level of cytotoxicity must be considered. Higher transfection efficiency contributes to greater success. However, low toxicity methods cannot be ignored, because highly cytotoxic methods can cause adverse effects in the form of visible morphological changes and unknown changes in gene expression or stress response pathways.

Transfection classification and application

Transfection is used to study the function and regulation of genes or gene products, to produce genetically modified organisms, and is used as a powerful analysis tool for gene therapy methods. According to the time range of the desired experiment and the goal, transfection can be divided into transient transfection and stable transfection.

  • Transient transfection
  • Transiently transfected cells are usually harvested within 24-96 hours after transfection. They are usually used to study the short-term expression of genes or gene products, perform RNA interference (RNAi)-mediated gene silencing, or rapidly-produce recombinant proteins.

  • Stable transfection
  • In order to obtain long-term gene expression, the DNA vector needs to be integrated into the host chromosome. This process requires selective screening and clonal isolation. It is usually used for long-term pharmacological research, gene therapy, or long-term genetic regulation mechanism research.