Introduction to Transfection

Image
solutions_feature_gxt4_transfection.jpg

Transfection generally refers to the introduction of foreign DNA into bacterial and/or mammalian cells. Transfection is an important tool used in studies investigating gene function and the modulation of gene expression, thus contributing to the advancement of basic cellular research, drug discovery, and target validation. This section provides an overview of different transfection methods, transfection workflow, factors affecting transfection efficient, and protocols.

Related Topics: Posttransfection Analysis of Cells, Instrument-Based Transfection Methods, and Chemical and Viral Transfection.

Page Contents
 
Transfection Methods

Transfection can be accomplished using chemical, biological, or physical methods. Common methods include electroporation, the use of a virus vector, lipofection, and biolistics. Many types of genetic material, including plasmid DNA, siRNA, proteins, dyes, and antibodies, may be transfected using any of these methods. However, a single method cannot be applied to all types of cells; transfection efficiencies and cytotoxicity may vary dramatically and depend on the method, cell type being utilized, and types of experiments being performed. Therefore, to obtain high efficiencies, all relevant factors should be considered for planning and selecting the appropriate transfection method.

Method Function Recommended Cells Products
Electroporation Nucleic acids or other molecules are introduced into cells by creating transient pores in the plasma membrane using an electric pulse Eukaryotic cells (primary, stem cells), prokaryotic cells (bacteria, yeast), plant protoplasts Gene Pulser Xcell™ electroporation system

Gene Pulser MXcell™ electroporation system

MicroPulser™ electroporator
Lipid-mediated Uses lipids to cause a cell to absorb nucleic acids; transfer of genetic material into the cell takes place 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
Biolistic particle delivery Delivery of nucleic acids into cells via high velocity nucleic acid-coated microparticles Plant, primary cells, tissue, and in vivo applications Helios™ gene gun

PDS-1000/He™ biolistic particle delivery system
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  
 
Transfection Efficiency

Successful transfection is usually measured in terms of transfection efficiency and cell viability — the higher the efficiency and viability, the better the transfection. Several important factors, such as the DNA quantity and quality, cell type, cell health, and transfection method (as stated above) affect transfection results. Transfection efficiency, for example, varies greatly with the cell type and its physiological condition prior to transfection. Ideally, the cells should be actively growing, healthy, and free of contamination.

Another way to present factors impacting transfection results is to consider the entire transfection workflow and the series of key sub-experiments it comprises. Each sub-experiment can significantly affect efficiency and viability. The typical workflow for a transfection experiment is as follows:

Transfection Workflow

Typical workflow for a transfection experiment.

The following table summarizes the factors to consider for efficient transfection:

Cell Health

Cells should be grown in medium appropriate for the cell line, supplemented with serum or growth factors as needed for viability

  • Contaminated cells and media (e.g., contaminated with yeast or Mycoplasma) should never be used for transfection
  • Make sure the medium is fresh if any components are chemically unstable, e.g., thiamine
  • Medium lacking necessary factors, such as serum, can negatively affect cell growth
  • Incubate cells at 37°C supplied with CO2 at the correct percentage (5–10%) and kept at 100% relative humidity
Confluency

Transfect cells at 40–80% confluency (cell type dependent)

  • Too few cells will cause cell cultures to grow poorly without cell-to-cell contact
  • Too many cells results in contact inhibition, making cells resistant to the uptake of DNA and other macromolecules
  • Actively dividing cells take up DNA better than quiescent cells
Passages of DNA

Number of Passages (cell type dependent)

  • Number of passages should be low (<50)
  • Number of passages for cells used in a variety of experiments should be consistent
  • Cell characteristics can change over time with immortalized cell lines and cells may not respond to the same transfection conditions.
  • Cells may not respond to the same transfection conditions after repeated passages

DNA Quality and Quantity

  • Use high-quality plasmid DNA for transfections that is free of proteins, RNA, and chemicals
  • DNA is typically suspended in sterile water or TE buffer to a final concentration of 0.2–1 mg/ml
  • The optimal amount of DNA to use in the transfection will vary widely and depend on the type of DNA, transfection reagent/method, target cell line, and number of cells
Time/Serum

Time

  • Optimal transfection time depends on the cell line, transfection method, and molecule transfected
  • Transfection times vary from 30 min to 4 hr (or may require overnight incubation based on the reagent used); some reagents do not require either media changes or additions
  • Cell morphology is monitored during the transfection interval because some cell lines lose viability during this period, e.g., cells maintained in serum-free medium
  • In addition to saving time, a shortened transfection time may significantly reduce the risk of cell death during the transfection procedure

Serum

  • Transfection protocols often require serum-free conditions for optimal performance because serum can interfere with many commercially available transfection reagents
 
Transfection Protocol Library

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.

 
Further Reading

Belyansteva IA (2009). Helios Gene Gun-mediated transfection of the inner ear sensory epithelium. Methods Mol Biol 493, 103–123. PMID: 18839344

Fujiki R et al. (2009). GlcNAcylation of a histone methyltransferase in retinoic-acid-induced granulopoiesis. Nature 459, 455–459. PMID: 19377461

Helledie T et al. (2008). A simple and reliable electroporation method for human bone marrow mesenchymal stem cells. Stem Cells Dev 17, 837–848. PMID: 18752428

Hockemeyer D et al. (2009). Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol  27, 851–857. PMID: 19680244

Huang B et al. (2008). RNA interference-mediated in vivo silencing of fas ligand as a strategy for the enhancement of DNA vaccine potency. Hum Gene Ther 19, 763–773. PMID: 18627219

Shimamura K et al. (2007). Generation of secondary small interfering RNA in cell-autonomous and non-cell autonomous RNA silencing in tobacco. Plant Mol Biol  63, 803–813. PMID: 17225952

Su L et al. (2009). Neural stem cell differentiation is mediated by integrin beta4 in vitro. Int J Biochem Cell Biol 41, 916–924. PMID: 18834954

Tseng CN et al. (2013). A method to identify RNA A-to-I editing targets using I-specific cleavage and exon array analysis. Mol Cell Probes 7, 38–45. PMID: 22960667

Videos

Primary Cell Culture
The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation
This video protocol describes the preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation.
 
Gene Pulser Xcell™ Electroporation System: Components, Application, and Troubleshooting
This tutorial highlights the main components and features of the Gene Pulser Xcell system. It provides information about system installation and the setup of electroporation experiments, including important troubleshooting tips and answers to frequently asked questions. Ordering information for system components and accessories is also provided.
 

Documents MADHA

Number Description Options
5448
MicroPulser Electroporator Flier, Rev A
5542
Electroporation Systems Brochure, Rev A
[ Add to Cart (Free) ]
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
1908
Electroporation Cuvette Flier, Rev B
5553
Gene Modulation Workflow Brochure, Rev B
5554
Gene Silencing | RNAi Workflow Brochure, Rev B
1349
Electroporation of T-Cell and Macrophage Cell Lines
1365
Introducing Proteins Into Cells by Electroporation
1345
Electroporation of Primary Bone Marrow Cells
1355
Production of Hybridomas by Electrofusion
1345
Electroporation of Primary Bone Marrow Cells
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
2007
Bombardment-Mediated Transformation Methods for Barley
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
2453
Optimization of Gene Delivery Into Arabidopsis, Tobacco, and Birch Using the Helios Gene Gun System
1688
Optimization of Biolistic® Transformation Using the Helium-Driven PDS-1000/He System
1689
Comparison of Performance Characteristics of Different Biolistic® Devices
2768
Biolistic Gene Transfer to Generate Transgenic Schistosomes, Rev A
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
6176 Electroporation Systems Overview Click to download
6177 Biolistic Particle Delivery Systems Click to download
6178 Recommended Biolistic System by Cell Types Click to download
6179 Lipid Transfection Reagents Selection Guide Click to download