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Transfection
Transfection
• Transfection is the process by which nucleic acids
are introduced into mammalian cells
• Protocols and techniques vary widely and include
the following three methods; chemical-based transfection, non-chemical based
transfection, and particle based transfection
• 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 recovery, and target validation.
Chemical-Based Transfection Methods
Calcium Phosphate
• Combining positively charged calcium and negatively charged phosphate creates a
precipitate, which binds to DNA target sequence
Dendrimers
• Highly branched organic cationic compounds create a complex-mediated transfection, in
which DNA is bound electrostatically to dendrimers and taken up via endocytosis
Cationic Polymers
• Examples: DEAE-dextran or polyethylenimine
• Negatively charged DNA binds to the poly-cation, and taken up into the cell via endocytosis
Cationic Liposomes
• Inclusion of the DNA to be transfected in liposomes, which fuse with the cell
membrane and release the DNA into the cell
• How does this work?
• Positively charged head group of liposome interacts with the negative backbone of the DNA causing
encapsulation and interacts with the cell membrane to allow fusion
• Entrance facilitated by endocytosis
• Cationic lipid reagents differ from liposome reactions
• No encapsulation occurs
• Negatively charged DNA binds spontaneously to liposomes forming a complex
• Entrance facilitated by endocytosis
Non-Chemical Based Transfection Methods
Electroporation
• Physical transfection that uses electrical pulse to create temporary pores in cell
membranes
Cell Squeezing
• The formation of transient holes through shear forces altering cell shape
Sonoporation
• High-intensity ultrasound to induce pore formation in cell membrane
Optical Transfection
• A highly focused laser is responsible for creating a transient hole in the plasma
membrane
Particle-Based Methods
Gene Gun
• DNA is coupled into a nanoparticle of an
inert solid, which is then shot directly
into the target cell’s nucleus
Magnetofection
• DNA particles are associated with magnetic nanoparticles where application
of magnetic force drives the complex into the target cell
Microinjection
• Physically transferring material into the cytoplasm with a micropipette
Transient vs. Stable Transfection
Transient Transfection
• The introduced nucleic acid exists in the cell only for a limited period of time and is not
integrated into the genome
• Transiently transfected genetic material is no passed from generation to generation
during cell division, it will be lost by environmental factors or diluted during cell division
• However, high copy number of the transfected genetic material leads to high levels of
expressed protein within the period that it exists in the cell
Stable Transfection
• Long term introduction of DNA into cells
• Stable transfected genetic material will be passed from generation to generation during
cell division, due to the transfected DNA being incorporated into the genome
Research & Transfection
Types of Researcher's that would be doing transfection include:
Molecular Biologists
• Branch of science that is concerned with biological activity at the molecular level
• In particular, researching the coding sequences of DNA by transfecting Eukaryotic cells with
cis active elements of DNA and siRNA for gene silencing. This is used to manipulate genomic
DNA to determine the genetic makeup of certain traits
Bioinformatics Research Specialists
• The science of collecting and analyzing complex biological data such as genetic codes
• Specifically, these researchers are experimenting with optimizing gene transfection in
Murine Myeloma cells to determine the best transfection reagent to gain the most desirable
results
Research & Transfection
Biochemistry Researchers
• Biochemistry is the branch of science that explores the chemical processes within and
related to living organisms
• It focuses on what’s happening inside our cells, studying components like proteins, lipids
and organelles and looks at how cells communicate with each other
• Many have come up with new ways of transfecting a cell, including ‘Electric Shockmediated Transfection of cells: Characterization and optimization of electrical parameters.’
These biochemists are researching the exact amount of electrical shock to be given to cells
in order to create desired sized pours in the outer cell wall. The voltage produced
determines the different size pours, as well as determining the optimal electrical
conditions for DNA transfection.
Research & Transfection
Two specific Researchers in Canada who may be interested in the Mirus products includes;
McMaster University
• Location: Hamilton, Ontario
• Professor Ray Truant and a team of researchers are researching a solution to a longstanding medical mystery in Huntington’s disease (HD), which is a disease that causes an
increasing loss of brain cells at the center of the brain.
• Researchers are developing a new way to measure the shape of the huntingtin protein
inside a cell while it was still alive. Which is done through three single neurons transfected
with a disease-associated version of huntingtin protein. The results of this research will give
answers about HD
Research & Transfection
Health Canada Toxicology Division
• Location: Edmonton, Alberta: Rekha Mehta Ph.D. is the Chief in the toxicology Research
Division
• The toxicology topic being researched is Mutagenicity, which focuses on in vivo approaches
to studying the toxicology of food associated chemicals and examining the biological
mechanisms involved.
• The interest in Mirus products is due to the researchers using molecular biology techniques
to construct DNA vectors and performing DNA transfections in order to research
Mutagenicity.
• Specifically, this research department may be interested in TransIT® Transfection Reagents:
ideal for delivering nucleic acids to a variety of eukaryotic cells, as well as for in vivo
delivery in small animals. They provide high efficiency transfection with low cellular
toxicity.