PRINCIPLES AND METHODS OF TRANSFECTION IN ANIMALS

PRESENTED BY: ACHYUT BORAROLL NO :130209003SEMESTER: 5th sem.DEPT: BS MEDICAL BIOTECHGAUHATI UNIVERSITY, ASSAM

PRESENTATION OVERVIEWTRANSFECTION.TRANSFECTION VS TRANSFORMATION.PURPOSE OF TRANSFECTIONMETHODS OF TRANSFECTIONBACTERIAL TRANSFECTIONVIRAL TRANSFECTIONCHEMICAL TRANSFECTIONMECHANICAL TRANSFECTIONPHYSICAL TRANSFECTIONADVANTAGES AND DISADVANTAGES OF METHODS OF

TRANSFECTIONSUMMARY

TRANSFECTIONWhat is Transfection?Transfection is a method of transporting DNA, RNA and/or various macromolecules into an eukaryotic cell by using chemical, lipid or physical based methods.

Methods: (just a few examples…) Method Application

CaPO4, DEAE DNA Transfection Liposome Based DNA Transfection Polyamine Based DNA Transfection

Terminology :

Transfectants : Cells that have incorporated foreign DNA.

a) Stable : Integrated foreign DNA.b) Transient: Does not integrate foreign DNA, but genes are expressed briefly.

Transfection vs. TransformationCloning (uptake of genetic material) Cancer

Transformation Transfection Transformation

In unicellular organisms like prokaryotes (bacteria) or unicellular eukaryotes (amoeba)

In Metazoan Eukaryotic Cells

Advancement of a metazoan eukaryotic cell from being non-cancerous to cancerous.

Purpose/uses of Transfection

Study gene function and regulation.

e.g Antibody, vaccine, hormones.

Study protein expression.

Transfer DNA into embryonic stem cells.

Transfection

Non- Viral

Mechanical1.

Microinjection

2. Particle Bombardme

nt3. Single walled

Carbon nano-tubes.

Physical

1. Electroporat

ion2.Ultrasoun

d3.

Dendrimers

Biological/Viral

1.Bacterial2. Viruses

3. Virus Like Particles

1.Calcium phosphate method.2. Transfection with polyplexes and3.Liposomes and lipoplexes.

Chemical

Bacterial vectors for gene transfer The exploitation of living bacteria for gene transfer is central to the genetic manipulation of plants.

Agrobacterium tumefaciens and its close relatives have been used for 20 years to generate transgenic plants.

Recently, A. tumefaciens has been used to transfer DNA to human cells. The protoplast fusion technique can

be considered as highly efficient form of bactofection. However, this type needs human intervention, while the A. tumefaciens does not require human intervention.

How does the bacterial transfer of DNA happen?

The bacteria invades the host animal cells and undergo lysis within them releasing their plasmid DNA. Example of these bacteria including Salmonella species (lysis occurs in the phagocytic vesicle), Listeria monocytogenes and Shigella flexneri (lysis occurs for these two species after they escape from the vesicle).

The plasmid DNA then finds its way to the nucleus where it gets incorporated with the cell’s genome and gets expressed.

Contrary to the above mentioned bacteria, A. tumefaciens does not invade the cell, instead, it attaches

itself to the outside surface followed by conjugation.

BACTOFECTION

Viruses That are used as gene transfer vectors Virus particles have a natural ability to adsorb to the surface of the cells and gain entry. This

can be exploited to deliver recombinant DNA into animal cells.

Several classes of viruses has been used for gene therapy and at least 8 has been used in clinical trials. Transgenes may be incorporated into viral vectors either by addition to the whole genome or by replacing one or more viral genes. This can be done by ligation or by homologous recombination.

If the transgene replaces a none essential gene the vector is described as helper-independent .

If it replaces an indispensable gene, then this vector will be helper dependent. It is generally recommended to use vectors from which all viral coding sequences has been deleted such vectors are described as fully deleted or gutted or gutless vectors. These vectors contain just the cis-acting elements required for packaging and for genome replication.

Advantages of such vectors high capacity for foreign DNA, example, icosahedral viruses. because no viral gene products are made, the vector has no intrinsic cytotoxic effects.

Viruses in useViral Vector DNA Insert Size Expression Pitfalls

Retro viral 8 kb Stable Random insertion site

Lenti viral 9 kb Stable Random insertion site

Adeno Virus 8 kb Transient Highly immunogenic

Adeno associated Virus 5 kb Stable, site specific location

Requires helper virus and difficult to remove

Herpes Simplex Virus 30-40 kb Transient No gene expression during latent infection

Vaccina Virus 25 kb Transient Potential cytopathic effects

Adeno virus

These are viruses with a linear, double stranded genome, of approximately 36 kb. These are used frequently due to certain advantages;

stability high capacity for foreign DNA wide host range including none-dividing cells Ability to produce high titer stocks up to 1011 pfu/ml.

Adeno-associate virus (AAV) These viruses are not genetically related to adenovirus but is so-called because it was first

discovered as a contaminant in an adenovirus isolate.

The AAV is a single stranded DNA and is a member of the parvovirus family.

It is naturally replicating deficient, thus it requires the presence of another virus such as adenovirus to complete its infection cycle. AAV replicates lytically and produces thousands of progeny virions.

The dependence of AAV on a heterologous helper virus such as adeno virus provides an unusual degree of control over vector replication making AAV one of the safest vectors to use for gene therapy.

Other advantages of this viral vector is the wide host range that it exhibits including none dividing cells.

The AAV genome is small (5 kb) and comprises a central region containing rep (replicase) and cap (capsid) genes flanked by 145 kb inverted terminal repeats.

Foreign DNA replaces the cap region and gets expressed by indigenous promoter. However rep proteins might interfere with the expression process thus responsible for some of the cytotoxic effects of the virus.

New vectors are designed with the deletion of the rep and cap genes and only utilizing the repetitive sequence which are the only elements required for replication, transcription and proviral integration.

AAV vectors have been used to introduce genes efficiently into many cells including liver, muscle, and neurons.

ADENO ASSOCIATED VIRUS VECTOR TRANSFECTION

Retrovirus-mediated gene transfer It involves the deletion of essential genes gag, pol, and env from retrovirus which make it replication deficient. and then insert the transfected gene.

Virus like particles(vlp)

Alternative approach to classical methods

Viral capsid- without viral genetic information.Eg: Pappilloma viruses: L1 and L2 proteins.

Predominantly use – vaccinationGene delivery – human polymo JC virus, murine polymovirus, pappilomaviruses and AAV- based VLPs.

Isolation and purification of viral capsid proteins

Empty viral particles reconstituted and stored at -80 0C

Packaging with DNA or siRNA inside empty viral particles.

Uses of VLP

Chemical transfection

DNA will be taken from the surroundings when the DNA is presented as a synthetic complex either as, 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.

Chemical Transfection TechniquesCalcium phosphate method;

Involves the formation of a fine DNA/calcium phosphate co-precipitate which first settles on the cells and then internalized by endocytosis.

The precipitate must be formed freshly at the time of transfection. The DNA escapes and reaches the nucleus and can be then expressed. Since the cells must be coated by the calcium complex, monolayers of cells must be used for maximum efficiency. However, this method gives only 1-2% transfection efficiency.

Transfection with polyplexes;

This is more efficient than that with calcium as it gives more uniform particle size.

Polyplexes are a polycationic compounds that form soluble complexes (polyplexes) through spontaneous

electrostatic interaction with DNA.This method is adapted as a plasmid DNA transfer. The

efficiency of the method can be increased by exposing cells to osmotic shock or treatment of cells with chloroquine.

Anew generation of the polycationic compounds has been developed such as poly-L-lysine and synthetic poly amines, polyethelylenimines and dendrimers highly (complex molecules built in layers from a central initiator such as ammonia or ethylenediamine).

POLYPLEXES TRANSFECTION

Transfection with liposomes and lipoplexes

This can be done by packaging the DNA inside a fusogenic phospholipid vesicle which interacts with the target cell membrane and facilitate DNA uptake.

Briefly, bacterial cells will be transformed with suitable plasmid vector

and treated with chloramphenicol to amplify plasmid copy number. The cells will be treated with lysozyme to remove cell wall, resulting

in protoplasts that will be centrifuged gently onto a monolayer of mammalian cells to promote fusion among them using polyethylene glycol.

Using lyposomes is more commonly used for this type of transfection. This method is commonly known as lipofection. This method is far more efficient than chemical transfection method. With this method up to 90% of cells in culture dish can be transected.

Liposomes and lipoplexes.

Mechanical transfection

In this method, naked DNA is deposited directly into the cell by exploiting a mechanical force. It includes

1. Microinjection2. Particle Bombardment

3. Single walled Carbon nano-tubes.

Direct Microinjection:

Use of a fine needle. Has been used for transfer of DNA into embryonic stem cells.

Direct transfer of DNA into the cell without a carrier is called DNA microinjectin. This can only be done for only few cells at a time. This technique is used mainly for large cells such as oocytes, eggs and the cells of early embryos. The DNA can be directly injected into tissues, such as skin, muscle or internal organs or it can be injected into the blood.

Biolistic Particle delivery: Uses high-velocity for delivery of nucleic acids and penetration of cell wall.

Particle bombardment is another direct delivery method initially developed for the transformation of plants. This method involves coating small metal particles with DNA and accelerating them into target tissues using a powerful force such as the blast of high pressure gas or an electric discharge through a water droplet. In animals, this method is used for tissues such as skin cells in vivo rather than cultured cells.

Single walled carbon nanotubes – mechanical method

Unidimensional layer of carbon-hexagons form a tube. Functionalized- amino or carboxyl group Covalent or non-covalent bond with biomolecules. Diameter: 1-5nm; Length: 50-200nm Success: In vivo siRNA delivery.

Swnt

Physical transfection methods In this method, naked DNA is deposited directly into the cell by exploiting a physical

force. This includes;1. Electroporation

2.Ultrasound 3. DendrimersElectroporation

Is a physical transfection technique involves creating transient nano-meter size pores in the cell membrane by exposing cells to a brief pulse of electricity. The most critical parameter is the intensity and duration of electrical pulse.

Electroporation can be used for in vivo gene transfer particularly for surface or near surface tissue such as skin, muscle and certain tumors or even internal tissues such as liver. This can be achieved by direct application of electrodes to the skin following shaving and mild abrasion with the DNA being injected into the skin before electroporation.

Ultrasound transfection Involves the exposure of cells to a rapidly oscillating probe such as the tip of sonicator. In this

method the application of ultrasound waves to a dish or cells or a particular tissue results in the formation and collapse of bubbles in the liquid, including the cell membrane, a process known as cavitation.

The transient appearance of such cavities allows the DNA to cross the membrane into the cytoplasm. This method can be used both for in vivo or in vitro as the plasmid DNA is left structurally intact. As for electroporation, the DNA will be injected and then the ultrasound will be applied.

Pamam dendrimers- physical method

Polyamidoamine (PAMAM)- non-linear polycationic cascade- binds plasmid DNA

Activated PAMAM + plasmid DNA = condensation of nucleic acid Compact transfection complex – adhere to cell surface and taken up by

endocytosis Generation 6/7 with 6 & 10nm – gene transfer Commercially available – SuperFect transfection reagent and PolyFect

transfection reagent (QIAGEN, Germany)

Advantages/Disadvantages

Advantages: Provides the ability to transfer

in negatively charged molecules into cells with a negatively charged membrane.

Liposome-mediated transport of DNA has high efficiency. Good for long-term studies requiring incorporation of genetic material into the chromosome.

Disadvantages:

Chemical Reagents: not useful for long-term studies.

Transfection efficiency is dependent on cell health, DNA quality, DNA quantity, confluency (40-80%)

Direct Microinjection and Biolistic Particle delivery is an expensive and at times a difficult method.

Summary Chemical methods : system needs to be adapted to the cargo to be delivered. No separate genetic protocols for siRNA and plasmid DNA delivery. Physical methods : cytotoxicity, cellular uptake insufficient mostly.What is needed?

Specific tailor-made DNA and siRNA delivery systems Nucleic acid-based therapeutics : individualized medicines for specific

disease variation.The ideal method should have High transfection efficiency Low cell toxicity Minimal effects on normal physiology Be easy to use reproducible.

…dermal patches consisting of gene-transfected cultured skin, which secrete endogenous antimicrobial peptides such as B-defensins instead of exogenous antibiotics, can be a new DDS for the treatment of severe burns without decrease in cell viability.”

Another interesting use

Thank you…Do you think we have learned

some thing new?