Instrument-Based Transfection Methods

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Overview

Transfection of cells can be accomplished by various methods, including chemical, biological, and instrument-based. This section provides an overview of the different instrument-based transfection methods available, discusses how they work, and describes their pros and cons.

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

Overview of Instrument-Based Transfection Methods

Transfection can be accomplished using chemical, biological, or physical methods. Common methods include electroporation, the use of virus vectors, 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, no single method can 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 in planning and selecting the appropriate transfection method.

Method Function Pros Cons Cells Products
Electroporation Nucleic acids or other molecules are introduced into cells by creating transient pores in the plasma membrane using an electric pulse Nonchemical method that doesn't seem to alter the biological structure or function of the target cells

Easy to perform

High efficiency

Can be applied to a wide range of cell types
Cell mortality (if using suboptimal conditions) Eukaryotic cells (primary, stem cells)

Prokaryotic cells (bacteria, yeast)

Plant protoplasts
Gene Pulser Xcell electroporation system

Gene Pulser MXcell electroporation system

MicroPulser electroporator
Biolistic particle delivery Delivery of nucleic acids into cells via high-velocity nucleic acid-coated microparticles Simple, rapid, versatile technique

Targeted intracellular gene delivery

Cell type independent

Uses small amounts of DNA

Delivers single or multiple genes

No carrier DNA needed

Can deliver large DNA fragments

No extraneous genes or proteins delivered

Requires little manipulation of cells

High reproducibility
Generally lower efficiency compared to electroporation or viral or lipid mediated transfection

Limited bacterial transfection data

Requires the preparation of microparticles

Instrument cost

Requires purchase agreement
Plant

Primary cells

Tissue

In vivo applications
Helios® gene gun system

PDS-1000/He and Hepta systems
Microinjection Direct injection of naked DNA Can be used for many animals Laborious (one cell at a time)

Technically demanding and costly
Eukaryotic cells  
Laserfection/
optoinjection
Uses laser light to transiently permeabilize a large number of cells in a very short time Very efficient

Works with many cell types

Few cell manipulations needed

Requires cell to be attached

Expensive laser equipment required
Attached cells  

Electroporation

  • 1. Electroporation exposes a cell to a high-intensity electric field that temporarily destabilizes the membrane
  • 2. During this time the membrane is highly permeable to exogenous molecules present in the surrounding media
  • 3. DNA then moves into the cell through these holes
  • 4. When the field is turned off, the pores in the membrane reseal, enclosing the DNA inside

Electroporation of cells.

Biolistic Particle Delivery

Biolistics is the delivery of nucleic acids into cells by firing nucleic acid-coated microparticles into them.

Helios Gene Gun

  • For in situ, in vivo and in vitro transformations
  • Applications for animals, plants, cell culture, nematodes, yeast and bacteria
  • Pressure range 100–600 psi enables fine-tuning of penetration
  • Highly portable can be used in the field
  • Small target area for accurate targeting

Biolistic particle delivery workflow.

PDS-1000/He Biolistic Particle Delivery System

  • For in vitro, ex vivo (and in vivo for some plants and microbes)
  • Applications for animal cell and organ culture, plant cell culture and explants, pollen, insects, algae, fungi and bacteria
  • Pressure range 450–2200 psi gives flexibility and penetration — ideal for plant applications
  • Large target area — more cells can be transformed

Procedure

  1. DNA-coated microcarriers (thin plastic disk) are spread over the central area of that disk using a pipette tip.
  2. Disk loaded with the DNA-coated particles is placed into a holder inside the PDS-1000 system.
  3. The system uses high pressure helium, released by a rupture disk, and a partial vacuum, to propel the macrocarrier sheet loaded with DNA-coated gold macrocarriers toward the target cells.
  4. Macrocarrier is halted after a short distance by a stopping screen.
  5. DNA-coated particles continue traveling toward the target to penetrate the cells.
  6. Sample chamber is subjected to a partial vacuum, from 15 to 29 in. of mercury, depending on the target cells.

 

Workflow for delivery using PDS-1000/He system.

Microinjection

  • Direct injection of naked DNA
  • Laborious (one cell at a time)
  • Technically demanding and costly
  • Can be used for many animals

Microinjection of particles.

Laserfection/Optoinjection

  • This procedure uses laser light to transiently permeabilize a large number of cells in a very short time
  • Various substances, including ions, small molecules, dextrans, short interfering RNAs (siRNAs), plasmids, proteins, and semiconductor nanocrystals can be efficiently optoinjected into numerous cell types

Workflow for laserfection.

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

Benediktsson AM et al. (2005). Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures. J Neurosci Methods 141, 41–53. PMID: 15585287

Eizema K et al. (2000). Endothelin-1 responsiveness of a 1.4 kb phospholamban promoter fragment in rat cardiomyocytes transfected by the gene gun. J Mol Cell Cardiol 32, 311–321. PMID: 10722806

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

Gildea JJ et al. (2009). Caveolin-1 and dopamine-mediated internalization of NaKATPase in human renal proximal tubule cells. Hypertension 54, 1070–1076. PMID: 19752292

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

Wirth MJ and Wahle P (2003). Biolistic transfection of organotypic cultures of rat visual cortex using a handheld device. J Neurosci Methods 125, 45–54. PMID: 12763229

Zhang G and Selzer ME (2001). In vivo transfection of lamprey brain neurons by gene gun delivery of DNA. Exp Neurol 167, 304–311. PMID: 11161618

Related Content

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.
 
Literature
Number Description Download
0108 Simple and Rapid Optimization With Maximum Transfection Efficiency, Rev A Click to download
1345 Electroporation of Primary Bone Marrow Cells Click to download
1349 Electroporation of T-Cell and Macrophage Cell Lines Click to download
1355 Production of Hybridomas by Electrofusion Click to download
1365 Introducing Proteins Into Cells by Electroporation Click to download
1688 Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System Click to download
1689 Comparison of Performance Characteristics of Different Biolistic Devices Click to download
1908 Electroporation Cuvette Flier, Rev B Click to download
2007 Bombardment-Mediated Transformation Methods for Barley Click to download
2015 Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cell Types Click to download
2051 Transformation of Filamentous Fungi by Microprojectile Bombardment Click to download
2087 Biolistic Transfection of Organotypic Brain Slices and Dissociated Cells Click to download
2433 Transformation of Nematodes With the Helios Gene Gun Click to download
2453 Optimization of Gene Delivery Into Arabidopsis, Tobacco, and Birch Using the Helios Gene Gun System Click to download
2497 Biolistic Purchase Demonstration Agreement, Rev E Click to download
2531 Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun Click to download
2552 The Gene Gun: Current Applications in Cutaneous Gene Therapy, Rev A Click to download
2658 Single-Cell Complementation of Barley mlo Mutants Using a PDS-1000/He Hepta System Click to download
2726 Delivery of pCMV-S DNA Using the Helios Gene Gun System Is Superior to Intramuscular Injection in Balb/c Mice Click to download
2768 Biolistic Gene Transfer to Generate Transgenic Schistosomes, Rev A Click to download
5399 Gene Pulser siRNA Electroporation References, Rev A Click to download
5443 Biolistic Particle Delivery Systems Brochure, Rev A Click to download
5445 Gene Pulser Xcell Electroporation System Flier, Rev A Click to download
5446 Helios Gene Gun System Flier, Rev A Click to download
5447 Biolistic PDS-1000/He System Flier, Rev A Click to download
5448 MicroPulser Electroporator Flier, Rev A Click to download
5542 Electroporation Systems Brochure, Rev A Click to download
5554 Gene Silencing | RNAi Workflow Brochure, Rev B Click to download
5555 Protein Interaction Analysis Workflow Brochure, Rev B Click to download
5582 Gene Pulser Electroporation Buffer Product Information Sheet, Rev A Click to download
5598 Gene Pulser MXcell Electroporation System Flier, Rev B Click to download
5603 Optimization of Electroporation Using Gene Pulser Electroporation Buffer and the Gene Pulser MXcell Electroporation System, Rev A Click to download
5622 Optimization of Electroporation Conditions With the Gene Pulser MXcell Electroporation System, Rev A Click to download
5634 Gene Pulser MXcell Electroporation System Brochure, Rev A Click to download
5641 The Gene Pulser MXcell Electroporation System Delivers Consistent Results Required for Optimizing Delivery Protocols, Rev A Click to download
5684 Poster: Optimization Is Essential to Increase Transfection Efficiency and Cell Viability in Primary and Difficult-to-Transfect Cell Lines, Rev A Click to download
5686 Optimization of Electroporation Conditions for Jurkat Cells Using the Gene Pulser MXcell Electroporation System, Rev A Click to download
5687 Transfection of Mammalian Cells Using Preset Protocols on the Gene Pulser MXcell Electroporation System, Rev A Click to download
5704 Electroporation Conditions for Chinese Hamster Ovary Cells Using the Gene Pulser MXcell Electroporation System, Rev A Click to download
5720 Transfection of Neuroblastoma Cell Lines Using the Gene Pulser MXcell Electroporation System, Rev A Click to download
5733 Transfection of Chinese Hamster Ovary-Derived DG44 Cells Using the Gene Pulser MXcell Electroporation System, Rev A Click to download
5774 Delivery of siRNA by Electroporation Into Primary Human Neutrophils Using the Gene Pulser MXcell System, Rev A Click to download
5778 Electroporation Parameters for Transfection of HL-60 Leukocytic Cell Line With siRNA Using the Gene Pulser MXcell System, Rev B Click to download
5823 Electroporation of Primary Murine Mast Cells Using the Gene Pulser MXcell Electroporation System, Rev A Click to download
5842 Optimization of Electroporation Conditions for Two Different Burkitt Lymphoma Cell Lines Using the Gene Pulser MXcell System, Rev B Click to download
5858 The Gene Pulser MXcell Electroporation System Provides Reproducible Results in Electroporation Plates and Cuvettes With the Same Protocol, Rev A Click to download
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 Click to download
5904 Transfection of Mouse and Human Embryonic Stem Cells by Electroporation Using the Gene Pulser MXcell System, Rev A Click to download
5924 Stem Cell Basics for Life Science Researchers Brochure, Rev A Click to download
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
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