transfection method
TRANSCRIPT
Transfection method
Nasim Arshadi
process of deliberately introducing nucleic acids into cells.
often used for non-viral methods in eukaryotic cells.
Cells that have incorporated the foreign DNA are called
transfectants.
Terminology
Transfection: this term is reserved to describe nonviral gene delivery method in eukariotic in animal cell.
Transformation: this term is reserved to describe nonviral gene delivery method in bacteria and nonanimal eukaryotic cell
Transduction or infecion :is a reserved to define gene delivery method using viruses.
Type of transfection
Transient transfectionwhen DNA is not integrated into host genome but genes are expressed for a limited time(24-96 hours(.the effect on target gene expression is temporary (24-72 hours for RNA probes, 48-96 hours for DNA probes).
used for studying: gene knockdown or silencing with inhibitory RNAs, or protein production on a small scale
Stable or permanent transfection
transfected genetic material is integrated into the host cell genome. used for studying: protein production on a large scale, longer-term pharmacology studies, gene therapy or research on the mechanisms of long-term genetic regulation.
Cell type Cell health Confluency Serum time DNA quality & quantity
Factors Influencing Transfection Efficiency
TRANSFECTION
WORKFLOW
Tissue culture count cells
Resuspend cells in electroporation
buffer
Add nucleic acid
Transfect cells
Plate cells
Analysis
Microscopy
Gene ExpressionProtein Expression
Western blot analysis
Reporter gene
activity
Flow cytometry
Real time PCR
Common transfection
methods
• Electroporation• Microinjection• Biolistic Particle Delivery• Laserfection/optoinjection
Instrument(physical)
based method
• Cationic Lipids : Liposome/Lipoplexes• Calcium phosphate• Cationic polymer• DEAE-Dextran• Magnet-mediated transfection• Activated dendrimers
Reagent (Chemical)-
based method
• Viral methodBiological
based method
a complex with overall positive charge, allowing it to interact with negatively charge cell membrane and promote uptake by endocytosis
as lipophilic complex that fuses with the cell membrane and deposits the transgene directly into the cytoplasm
Electrostatic interactions between the positive charges of
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.
the cationic lipid is mixed with a neutral lipid such as L-dioleoylphosphatidylethanolamine(DOPE)which can enhance the gene transfer ability of certain synthetic cationic lipids
Cationic Lipids
The successful use of liposome compounds is dependent upon several factors including lipid formulation, particle size and the method of liposome preparation. An overall net positive charge of the complex allows
closer association of the complex with the negatively charged cell membrane.
Entry of the liposome complex into the cell:endocytosis or fusion with the plasma membrane via the lipid moieties of the liposome
When liposomes encounter nucleic acids they re-form into nucleic acid lipid complexes called lipoplexes which can be actively taken up by eukaryotic cells by means of endocytosis
Advantages of lipids: Deliver nucleic acids with high efficiency Easy to use, minimal steps required. Works in a wide variety of eukaryotic cells can be used for in vivo transfer of DNA and RNA to animals
and humans successful delivery of DNA of all sizes from oligonucleotides to
yeast artificial chromosomes Disadvantages of lipids: Not applicable to all cell types. low efficiencies in most primary cells
Pros and conscationic lipid
Principle DNA is mixed with calcium chloride Addition to buffered saline/phosphate solution and incubating at
room temperature Formation of DNA-calcium phosphate coprecipitates which adhere to
surface of cells Uptake presumably by endocytosis or phagocytosis
Calcium phosphate
Advantages: easily available Inexpensive Can be applied to wide range of cell types Can be used for transient and stable transfection Calcium phosphate appears to provide protection against
intracellular and serum nucleases. Disadvantages: low efficiency is not suited for in vivo gene transfer to whole animals Size and quality of the precipitate are crucial to the success of
transfection toxicity, especially to primary cells its sensitivity to slight changes in pH, temperature and buffer salt
concentrations.
Pros and ConsCalcium phosphate
Cationic polymer differ from cationic lipid in that they do
not contain a hydrophobic moiety and are completely soluble in water.
Cationic polymer cationic polymer include: polybrene, polyethyleneimine(PEI) and
dendrimers.
ability of cationic polymer more efficiently condense DNA. cationic polymer cannot release their DNA load into the cytoplasm
Cationic polymer
synthesized (lengths , geomrtry)
Dendrimers are highly branched, globular macromolecules that are capable of interacting with DNA to form small complexes.
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.
Complexes bound to the cell surface are taken into the cell by nonspecific endocytosis.
Dendrimers
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.
Dendrimers are stable in biological liquids and are not sensitive to temperature. highly efficient tools for tissue culture transfections.
However, dendrimers are also The downside of dendrimers non-biodegradable and may cause significant cytotoxicity.
DEAE-dextran was the first non-viral transfection method verified by Vaheri and Pagano in 1965.
DEAE-Dextran is a cationic polymer that tightly associates with the negatively charges nucleic acids.
principle DNA is mixed with DEAE-dextran DNA/polymer complex comes into contact with
negatively charged membrane due to excess of positive charge contributed by polymer entry in to the cytoplasm via endocytosis or osmotic shock induced by DMSO or glycerol.
Uptake presumably by endocytosis
DEAE-Dextranchemical method
Advantages: Inexpensive Easy to perform and quick Can be applied to a wide range of cell types. Low cost Disadvantages: High concentration of DEAE-Dextran can be toxic to the
cell Transfection efficiency will vary with cell type Can be used only for transient transfection Typically produces less than 10% delivery in primery cells
Pros and ConsDEAE-Dextran
Magnet-mediated transfection
(Magnetofection) Magnet-mediated method
uses magnetic force to deliver DNA into the target cells.
Nucleic acids are first associated with magnetic nanoparticles.
DNA are bound to MNPs, which then move from the media to the cell surface by applying a magnetic force.
Plasmid DNA or siRNA is attached to magnet nanoparticles and incubated with cells in culture.(i) An oscillating magnet array below the surface of the cell culture
plate pulls the particle into contact with the cell membrane (ii) and drags the particles from side-to-side across the cells (iii)mechanically stimulating endocytosis (iv)Once the particle/DNA complex is endocytosed, proton sponge
effects rupture the endosome (v) releasing the DNA (vi)which then transcribes the target protein.
Advantages Rapid Increased transfection efficiency by direct transport,
especially for low amount of nucleic acid High transfection rates for adherent mammalian cell
lines and primary cell cultures Can also be performed in the presence of serum Disadvantages Require adherent cells; suspension cells need to be
immobilized or centrifuged.
Pros and consMagnet-mediated transfection
in this method, naked DNA is deposited directly into the cell
by exploiting a physical force
Instrument(Physical )-based method
Electroporation was first reported for gene transfer
studies in mouse cells . This technique is often used for cell types such as
plant protoplasts. Electroporation uses an electrical pulse to create
temporary pores in cell membranes through which substances like nucleic acids can pass into cells.
More modern instrumentation successful transfer of DNA and RNA to primary and stem cells.
Electroporation
ectr
opor
atio
n
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
Advantages fast doesn't seem to alter the biological structure or function of the
target cells Easy to perform High efficiency Can be applied to transient and stable transfection a wide range of
cell types
Disadvantages Cell mortality low efficiency in primary cells and high mortality rates, caused by
the high voltage pulses or only partially successful membrane repair.
Pros and conselectroporation
Cell squeezing is a method invented in 2012
by Armon Sharei, Robert Langer and Klavs Jensen.
Cell squeezing
It eliminates the possibility of toxicity or off-target effects as
it does not rely on exogenous materials or electrical fields. describe a microfluidic approach to delivery in which cells
are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter.
transient holes that enable the diffusion of material from the surrounding buffer into the cytosol.
The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells.
involves projecting microscopic heavy-metal particles (often gold or tungsten) coated with nucleic acids into recipient cells at high velocity using a biolistic device (i.e., “gene gun”).
Biolistic particle delivery can be used to transiently transfect dividing and non‑dividing cells in culture as well as cells in vivo, and it is often used for genetic vaccination and agriculture applications.
particle bombardment) ) Biolistic
ParticleDelivery
Delivery of nucleic acids into cells via high-velocity nucleic acid-coated microparticles
Helios Gene Gun
PDS-1000/He Biolistic
Particle Delivery System
Pros and cons
Biolistic particle deliveryAdvantages
Simple, rapid technique Cell type independent Uses small amounts of DNA Can deliver large DNA
fragments Requires little manipulation
of cells High reproducibility
Dis advantages Generally lower efficiency
compared to electroporation or viral or lipid mediated transfection
Requires the preparation of microparticles
Instrument cost causes physical damage to the
samples, and necessitates high cell numbers due to high mortality.
delivers nucleic acids into the cytoplasm or the nucleus one cell at a time by means of a fine needle;
therefore, this method is limited to ex vivo applications such as the transfer of genes into oocytes to engineer transgenic animals or the delivery of artificial chromosomes.
Microinjection
Advantage: Frequency of stable integration of DNA is far better as compare to
other methods.
Method is effective in transforming primary cells
The DNA injected in this process is subjected to less extensive modifications.
used to transfer DNA into embryonic stem cells that are used to produce transgenic organisms
Disadvantage: Costly.
Skilled personal required.
Method is useful for protoplasts and not for the walled cells.
Pros and cons
This procedure uses laser light to transiently
permeabilize a large number of cells in a very short time.
Various substances, including ions, small molecules, (siRNAs), plasmids, proteins, can be efficiently optoinjected into numerous cell types.
When the laser induces a pore in the membrane, the osmotic difference between the medium and the cytosol facilitates the entry of nucleic acids or other desired substances in the medium into the cell.
Laserfection/Optoinjection
Pros and cons
Advantage high transfection efficiency and the ability to
make pores at any location on the cell. Work with many cell type Fewer cell maniupullation neededDisadvantage requires an expensive laser-microscope system
and the cells to be attached to a substrate.
Comparison of Different Gene Transfer Methods
Comparison of Different Gene Transfer Methods
A Comparison of Different Viral Systems
