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TRANSFECTION
What is transfection ?
Principle
Vectors for delivery of DNA
Different approches
Two types of transfections
TRANSFECTION
What is transfection?
- Transfection is the transfer of DNA into cells.
- Transfection has played a role in :
- Aiding in gene cloning
- Analysis of gene expression
- Gene funtion
- Characterisation of DNA replication
- Recombinaison and repair
- It is a key technique for analysing mammalian cells.
TRANSFECTION
Principle :
1. Vectors for delivery of gene
2. Penetration in the nucleus
3. Transcription : mRNA synthesis
4. Translation : protein synthesis
TRANSFECTION
Vectors for delivery of DNA :
The repertoire of available techniques to deliver exogenous DNA into enkaryotic cells is very wide.
The field of vectors can be subdivised into several groups :
TRANSFECTION : CHEMICAL METHODS
The earliest transfection procedures utilized chemical methods, including calcium phosphate and
diethylaminoethyl (DEAE)–dextran methods.
1. Calcium phosphate coprecipitation : introduced by Graham and van der Eb in 1973.
- A solution containing calcium chloride and DNA are slowly mixed. This solution is layered on to
the cultured cells and is taken up by endocytosis.
- Transfections using this method is easy to perform.
- The efficiency of DNA transfer, is sensitive to experimental conditions such as calcium chloride
concentration, DNA concentration, and pH of the transfection solution.
- This method is appropriate for both transient and stable transfections.
2. DEAE–dextran method : DEAE–dextran and DNA
are mixed, and the solution is applied to cells.
- The efficiency of stable transfection is generally low.
- This method produces reliable results in transient transfection
experiments using adherent cells.
These chemical transfection methods are rarely used today. Many cell types,
including primary cell cultures, are difficult to transfect.
A number of alternative approaches offer a higher efficiency, lower
variability, and lower toxicity for a greater variety of cell types and have
largely replaced older chemical methods.
TRANSFECTION : CHEMICAL METHODS
3. Liposome-based Methods
Liposomes, complexes of synthetic lipids, are
popular reagents for transfection of DNA due to their
high efficiency and simplicity.
Most procedures use cationic (positively charged)
liposomes that bind the negatively charged phosphates
of the DNA molecules (Gao and Huang, 1995).
Mechanism of liposome gene transfer in
eukaryotic cells
When DNA is complexed with the liposome,
the positive charge is attracted to the negative charge of
the sialic acid residues on the surface of the cell.
The DNA–liposome complex is thought to enter the cell
via endocytosis. Most DNA goes to lysosomes and is
degraded, but a fraction enters the nucleus.
TRANSFECTION : MECANICAL METHODS
2. Particle bombardment, or the ‘gene gun’ approach (Yang et al., 1990).
- This method requires the precipitation of the DNA on gold particles.
-The DNA/ gold microcarriers are then loaded into a device that uses a force generated by
compressed helium expansion to achieve penetration of the DNA/gold into the cells.
- The gene gun has been used to deliver DNA :
- In vivo to targeted tissues in humans and animals
- In primary cultures from human tissues, including difficult-to-transfect neuronal cultures
- In neuronal slices
- The method is also effective in transfection of cell culture lines and can be used to generate stably
transfected cell lines.
- Like the other physical methods, it requires special equipment
TRANSFECTION : PHYSICAL METHOD
1. Electroporation :
This method is an increasingly popular physical method that relies on short pulses of
high voltage to carry DNA across the cell membrane. This shock is thought to cause temporary formation of pores in the cell membrane, allowing DNA molecules to pass through.
In vitro electroporation :
Cells are suspended in buffer and placed in an electroporation cuvette. The cuvette is
placed in an electroporation chamber, and voltage is applied. Nearly all cell types can be transfected in this manner .
Electroporation requires specialized equipment.
It is simple to perform electroporation and is often the best choice for cells that cannot be
transfected by other methods.In some cases, commercially available electroporation kits can be used, which have been optimized by the manufacturer for a particular cell type. Kits are often expensive.Although cell death may remain high, transfection efficiency is generally good for cell lines (50–80%)for primary cell cultures (30–70%)
TRANSFECTION : PHYSICAL METHOD
Pulse Generator EPI 2500 with square
wave pulses in the range of 0-2500V
- With the electroporation method, several variables can be optimized to provide acceptable :
- efficiency and toxicity
- DNA concentration
- electroporation buffer composition
- applied voltage
- duration of pulse
- All these parameters affect toxicity and transfection efficiency.
- These variables are often difficult to optimize
TRANSFECTION : PHYSICAL METHOD
2. Laser Poring
- Exposition of cells to short pulses of a laser beam.
- Small holes are transiently formed in the cell membranes that make it easier for DNA molecules suspended in the surrounding medium to enter
the cells.
TRANSFECTION : NOVEL METHODS
A number of novel methods have recently been developed for mammalian
cell transfection. Most of these methods claim improved efficiency,
reduced toxicity, or wider versatility
1. Polyamine dendrimers
Positively charged, highly branched molecules called ‘dendrimers’ complex with negatively charged DNA and are used in a manner similar to liposomes in transfection.
Dendrimers are reported to have decreased toxicity.
Commercially available dendrimers include SuperFect (Qiagen).
2. Magnetofection
Magnetofection™ is a novel, simple and highly efficient transfection method. This technology associates nucleic acids with cationic magnetic nanoparticles. The resulting molecular complexes are then transported into cells supported by an appropriated magnetic field.Three types of adapted and ready-to-use reagents are proposed :
- PolyMag, for all nucleic acid and all cell types
- SilenceMag, specifically designed for siRNA delivery
- CombiMag, unique solution for all vectors: transfection reagents and viruses
TRANSFECTION : BIOLOGICAL METHODS
1. Receptor-mediated gene transfer
- Receptor-mediated gene transfer DNA to be delivered into
mammalian cells is bound to a polylysine–transferrin
conjugate.
- Polylysine, a polycation, binds with DNA molecules and
condenses them into small complexes. The ligand
transferrin binds to its specific receptor located on the
external side of the cell membrane. Subsequently, DNA is
taken up into the cell by endocytosis
TRANSFECTION : BIOLOGICAL METHODS
2. Virus-mediated gene transfer
- Virus-mediated DNA delivery is based on the natural process by which viruses infect
mammalian cells.
- Viruses have specific mechanisms for the expression of their genomes, either through
maintenance of episomal elements or by integration into the host cell genome.
- Many viral vector systems are used for gene transfer, including retrovirus, SV40,
adenovirus, associated adenoviruses, vaccinia virus, herpes virus and several others.
TRANSFECTION : BIOLOGICAL METHODS
2.1. Retroviruses
- Retroviral vectors are used most frequently to introduce genes into
mammalian cells.
- A retrovirus-based gene transfer system consists of two components:
- the transfer vector, which harbours a foreign gene
linked to elements needed for retroviral replication
- the packaging cell, which supplies the necessary
retroviral proteins
Viral vectors mechanism to deliver their nucleic acid
- By its stable integration into a chromosome,
genetic material introduced by a retroviral
mechanism will remain in the body forever
unless the targeted cells and their progeny are
eliminated.
- This advantage concerns only divinding cells.
- Recently, the use of HIV-1 and other
lentiviruses showed that these vectors may
enter also nondividing cells
TRANSFECTION : BIOLOGICAL METHODS
2.2. Adenoviruses
Adenovirus vectors have proven able to mediate
long lasting expression in nondividing or slowly
dividing cells.
TRANSFECTION : different approches
Ex vivo :
- A sample of the patient`s cells can be removed and
exposed to the vector in a laboratory setting. The cells
containing the vector are then returned to the patient
In situ :
- The vector can be injected directly into a specific
tissue in the body, for example : tumor
In vivo :
- The vector can be injected intravenously (by IV)
TWO TYPES OF TRANSFECTIONS
1. Transient transfection
When the introduced DNA remains in the cell for a short time, independent of the chromosome.
2. Stable Transfection
For many experiments, short-term studies using transiently transfected plasmids are not
sufficient. It is often necessary to have the gene of interest integrated into the host chromosomes
for long-term analysis.
- Some vectors are used for transient transfection, others for stable transfection
Procedure :
To obtain stable transfection, we perform in well
plates according to the protocol for transfection in
adherent cells. Then, we start selection with
appropriate antibiotic 24–48 h after transfection.
The selectable marker is often a gene conferring
antibiotic resistance.
Cells expressing the gene can survive in antibioticcontaining
medium, while cells without the
integrated DNA will die.
DNA CLONING
What is DNA cloning ?
Restriction enzymes
DNA ligases
Types of vectors : plasmids
Reporter genes
Isolation of plasmid from E. Coli
Electrophoresis
Dosage of DNA
DNA CLONING
Whay is DNA Cloning ?
DNA cloning is the art of creating recombinant DNA molecules that can be :
- introduced into living cells
- replicated
- passed on the daughter cells as that cell divides
DNA cloning has been made possible by the discovery of two types of
protein :
- Restriction enzymes which break such that they have suitable termini
for ligation
- DNA ligases which are capable of ligating the molecules of DNA
DNA to be inserted
Restriction enzymes
DNA ligases
Recombinant plasmid
DNA CLONING
1. Restriction enzymes or endonucleases :
- Discovered in the late 1960.
- Recognize specific DNA sequences and make double-strand cleavages.
- Most restriction enzymes typically recognize DNA sites that are 4–8 base pairs in length.
- Bacterial species contain restriction–modification systems with genes that encode both a restriction endonuclease and a methyltransferase that recognizes the same sequence. The host DNA is fully protected from the action of the restriction enzyme by the methyltransferase
DNA CLONING
2. DNA Ligases
- DNA strands can be joined through the action of the enzyme DNA ligase.
- The normal biological role of the ligase, is to join the series of Okazaki fragments
during replication.
- « T4 DNA Ligase » is used in cloning experiment (E. Coli)
DNA ligases are able to connect between 3`-
OH carbone and phosphate-5` of two
neighboring nucleotides in the DNA strand.
DNA CLONING
3. 1. Types of vectors : Plasmids
- Plasmids are the most widely used vectors.
- It is very easy to isolate them from the host cell.
- They are extrachromosomal genetic elements which control their own replication.
- Bacterial plasmids are circular and double-stranded that range in size from 1 to 10 kb.
The fact that bacterial plasmids are nonessential for their host organism,
and have a size and form suitable for manipulation, has made possible
their widespread applicability to DNA cloning
DNA CLONING
3.1. Types of vectors : Plasmids
Promoter : to initiate high levels of transcription (T7)
Origin of replication : sequence reconigzed by host cell DNA replication systems.
X R Selection : gene that encode resistance to antibiotic
MCS : multicloning site, recognition sites for restriction enzymes (region where forein DNA is to be inserted
DNA CLONING
Aschematic representation of a typical cloning experiment.
(a) The vector is cut within its multicloning site (MCS).
(b) The target DNA is cut so as to produce termini compatible with the vector. ©The vector and insert are ligated to produce recombinant DNA.
(d, e) Recombinant DNA is introduced into appropriate host cells. In this illustration the vector encodes resistance to an antibiotic, X.
(f) If the cells are plated out on to medium containing X, only cells that have been transformed will grow and divide to form colonies
Isolation of Plasmid DNA from E. coli
Essentially all genes can be expressed by using recombinant methods. Gene expression in Escherichia coli is the most efficient system.
1. Transformation
- After the transformation, the bacteria are cultured in growth medium.
Since a plasmid usually harbours a selectable marker encoding an antibiotic overcoming protein, the corresponding antibiotic is added to the growth medium to maintain the plasmid in the growing bacterial cells. Coincident with bacterial growth, the plasmid DNAs are also replicated
Isolation of Plasmid DNA from E. coli
2. Principle to purify plasmid :
- Lysis of bacterial cells on modified SDS-alkaline
- Selective binding of the DNA to silica beads
- The yield of plasmid is up to and for midi-prep 100 μg and 500 μg maxi-prep
- The protocols provide a simple and reliable way for rapid isolation of plasmid.
- The high-quality plasmid DNA is eluted and recovered by ethanol precipitation.
Plasmid thus prepared can be used immediately for many plasmid routine applications such as
- DNA sequencing
- restriction digestion
- in vitro transcription
- library screening
- ligation
- transformation.
Microbiology laboratory
- Incubator (37°C)
- Temperature controlled Shaker
- Refrigerated centrifuge
- Electroporator
- Hood
- Ice
- Refrigirator
DNA CLONING : Electrophoresis
Why electrophoresis ?
- To verify if the purified plasmid obtained after the transformation is correct.
- Plasmid is digested using restriction enzyme
Principle of Gel Electrophoresis
- Electrophoresis is the migration of charged particles or molecules in an electric field. This
occurs when the substances are in aqueous solution.
- The speed of migration is dependent on the applied electric field strength and the charges of
the molecules.
Agarose gels are used as thick layers in flatbed chambers mainly for preparative purposes.
DNA CLONING : Electrophoresis
Electrophoresis setup
The bands are visualized with
fluorescent dyes that are visible
in UV light using ethidium
bromide.
Migration of deoxyribonucleic acid fragments
The relative mobilities of deoxyribonucleic acid
(DNA) molecules are dependent on the sizes of
the molecules.
DNA CLONING : Dosage of DNA
MOLECULAR BIOLOGY LABORATORY
- PCR System
- Centrifuge
- Hood
- Spectrophotometer
- Electrophoresis apparatus
- Generator with cuve
- UV transilluminator
UV transilluminator
APPLICATIONS
Transgenic models
Gene therapy
Antisense strategy
APPLICATIONS
Gene transfer technology in vitro is used for many purposes :
1. To produce large amounts of proteins
2. To evaluate the effects of specific mutations introduced in genes
3. To verify the identity of a cloned gene
4. To study the physiological consequences of overexpression of a
protein of interest
APPLICATIONS
2. Transgenic models :
Transgenic mice expressing Lac Z
Transgenic mice expressing
Growth hormon
APPLICATIONS
Transgenic mice expressing GFP
In transgenic animals, expression can be
monitored when a given tissue is subjected
to certain promoter-responsive stimuli
APPLICATIONS : Gene Therapy
- Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein.
- If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein.
- Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can`t cause disease when used in people.
- Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome
APPLICATIONS : Clinical trials
TYPES OF VECTORS PROTOCOLS
Retroviruses 50%
Adenoviruses 15%
Chemical vectors and DNA nude 20%
Others 15%
APPLICATIONS : Clinical trials
Approaches to Gene Therapy for Cancer
There are different approaches to gene therapy:
- Augmentation of immunotherapy
- Gene replacement therapy
- Drug sensitization
- Drug resistance
APPLICATIONS : Antisense strategy
Antisense Nucleic Acids in Biotechnology
- The strategy of antisense nucleic acid technology is to inhibit expression of a particular gene in a cell.
- This is achieved by the base pairing of a complementary
(antisense) oligo- or polynucleotide to a target RNA, which is usually a messenger RNA (mRNA).