Gene transfer is a technique to stably and efficiently introduce functional genes (that are usually cloned) into the target cells. Genetic transfer is the mechanism by which DNA is transferred from a donor to a recipient. Once donor DNA is inside the recipient, crossing over can occur. The result is a recombinant cell that has a genome different from either the donor or the recipient. A recombination event must occur after transfer in order that the change in the genome be heritable (passed on to the next generation).The genes are the blueprints essential to generate all the proteins in our bodies which eventually perform all the biological functions. Therefore, when a gene is efficiently introduced into a target cell of the host, the protein which is encoded by that gene is produced .
Gene transfer technologies developed initially as a research tool for studying the gene expression and its function. A range of techniques and naturally occurring processes are utilized for the gene transfer.
Techniques are available for the introduction of DNA into many different cell types in culture, either to study gene function and regulation or to produce large amounts of recombinant protein.
Cell cultures have therefore been used on a commercial scale to synthesize products such as antibodies, hormones, growth factors, cytokines and viral coat proteins for immunization.The most important application of this technology is in vivo gene therapy, i.e. the introduction of DNA into the cells of live animals in order to treat disease. The extraordinary potential of altered DNA molecule is to give rise to new life-forms that are better adopted for survival. Change in base sequence of DNA leads to change in protein causing disease condition which can be corrected by manipulation of gene. Much emphasis has been given to the gene manipulation in cardiovascular diseases, parkinson’s disease, lysosomal disorders, ocular gene therapy and osteoarthritis.
Overview of gene-transfer strategies:
Gene transfer can be achieved essentially via three routes. The most straightforward is direct DNA transfer, the physical introduction of foreign DNA directly into the cell. For example, in cultured cells this can be done by microinjection, whereas for cells in vivo direct transfer is often achieved by bombardment with tiny DNA-coated metal particles. The second route is termed transfection, and this encompasses a number of techniques, some chemical and some physical, which can be used to persuade cells to take up DNA from their surroundings. The third is to package the DNA inside an
animal virus, since viruses have evolved mechanisms to naturally infect cells and introduce their own nucleic acid. The transfer of foreign DNA into a cell by this route is termed transduction.
The choice of methods of DNA transfer depends upon the target cells in which transformation will be performed. It also depends upon the objectives of gene manipulation. The transfection may be either stable or transient. Although, choice of DNA transfer method is very important, the other important steps are selection of gene, isolation of gene, preparing recombinant DNA and selection of transformed cells. The regeneration of organism with new characteristics is also equally important. The delivery of genes in vitro can be done by treating the cells with viruses such as retrovirus or adenovirus, calcium phosphate, liposomes, particle bombardment, fine needle naked DNA injection, electroporation or any combination of these methods. These are the powerful tools for research and have possible applications in gene therapy.
FACTORS TO CONSIDER WHEN SELECTING A GENE DELIVERY METHOD
The advancement of new technologies has made gene delivery both more efficient yet more confusing given the formidable array of reagents and methods now available. Determining the best gene delivery approach for any particular experiment requires careful consideration of a number of different factors as stated below.
1. Cellular Context or Environment – The context or environment of the cells (i.e., in vivo vs. in
vitro vs. ex vivo) to which nucleic acids will be delivered is the first factor to consider when
deciding upon a gene transfer method. The obstacles and challenges posed by delivery to in
vitro cell cultures are very different than those posed by in vivo or ex vivo cells, and require
different tools and techniques for overcoming them. For example, targeting nucleic acids to
particular cells while avoiding other cells is usually a major concern for in vivo gene transfer
applications, sometimes a concern for ex vivo applications, but usually not a concern for in vitro
applications.
Another important issue that must be addressed when planning in vivo (but not in vitro) gene
delivery studies is the immunogenicity of the gene transfer system. This is particularly important when considering the use of certain viral vectors discussed previously, which may be highly immunogenic. Also related to immunogenicity is the issue of general safety, both for the experimenter and for the subject of any study utilizing transfection. Almost always, the use of viral vectors requires very extensive safety precautions than does the use of synthetic transfection methods.
2. Cell or Tissue Type – The origin of the cells or tissue to which nucleic acids will be
transferred is typically the second parameter considered when selecting a gene delivery
method. For a number of different reasons, the difficulty of cellular nucleic acid delivery varies
from cell type to cell type. Because of this, it is always important to review the literature to
determine if the cell type of interest has previously been utilized for gene transfer experiments,
which exact methods have been used, and what delivery efficiencies were achieved, i.e., what
percentages of cells actually transfected or transduced, with each method.
Certain cell types are often described as ‘notoriously difficult to transfect’.
These include primary cell cultures of many varieties. Successful gene transfer to such cells
usually requires a good deal of research before selecting any particular gene delivery method.
3. Delivery Efficiency – For most applications, transfecting or transducing as many cells as
possible is preferred, especially when it is necessary to detect the presence of rare transgene
products. Thus, it is important to determine the level of delivery efficiency required for a given
application before deciding on a particular gene transfer method. In most cases, it is found that
viral vectors provide the highest delivery efficiencies. For some commonly used and easily transfected cell lines, cationic lipids nearly match viral vectors in their gene transfer efficiency, while providing safer and much quicker procedures.
4. Stable vs. Transient Gene Delivery – Gene transfer procedures are frequently categorized
by whether the delivered gene remains separate from the host cell chromosome or whether it is integrated into the host cell chromosome. In the first case, known as transient gene delivery, expression of the transgene typically dissipates after a given period of time – usually within
several days – because the expression vector is either degraded or expelled from the host cell.
In the second case, known as stable gene delivery, expression of the transferred gene is
prolonged, or stable, because the vector is integrated into the host cell chromosome. Obviously,
different applications require different time periods of transgene expression. Thus, a careful
comparison between different gene delivery methodologies will allow a suitable choice for
generating the desired transient or stable expression.
In the case of viral vectors, determining whether a particular virus is suited for stable or transient
transduction is simply a matter of learning the general characteristics of the vector. In the case
of chemical or physical/mechanical gene transfer, determining whether a given method is
suitable for transient vs. stable delivery may be as easy as reading sales literature from the
manufacturer or checking primary research literature.
5. Type of Delivered Molecule – Another factor to consider when selecting a gene delivery
method is what type of nucleic acid or other molecule is being transfected or transduced. For
example, a reagent that efficiently delivers plasmids may not efficiently deliver DNA
oligonucleotides. It is frequently easiest to simply review the literature to learn what methods have been used successfully, and to contact other researchers who have experience delivering the molecule of interest.
6. Cytotoxicity – Another issue that comes up frequently when choosing among different gene
delivery methods is the cytotoxicity of the various methods and reagents. Some methods, such
as electroporation and gene guns, can be extremely harsh on cells and often result in the death
of a majority of cells. Also, the DEAE-dextran can be quite harsh on some cell types, especially
when a DMSO or glycerol shock step is used as part of the procedure. In some applications,
such as when a small number of transfectants are required from a recalcitrant cell type, this is
not a problem. In the majority of applications however, a high percentage of cell death is simply
not acceptable. The plethora of available cationic lipids and polymer reagents have varying
degrees of toxicity with different cell types, and their concentrations during transfection can be
optimized as needed to minimize cell death.
7. Suspension vs. Adherent cells – Most commonly transfected cells grow – and are
transfected – while attached to some type of support, whether that support is the tissue from
which they are derived, some inert membrane or scaffolding, or the surface of a tissue culture
plate. Less common are cells that grow while suspended in medium, such as those derived from
blood and other bodily fluids. Suspension cells are generally more difficult to transfect;
therefore, it may be necessary to rely on harsher gene delivery methods, such as
electroporation, gene guns, microinjection, or viral vectors for acceptable transfection levels.
Also, some cationic lipids can effectively transfect suspension cells, and should be tried when
possible due to their simplicity and cost-effectiveness.
8. Expertise – The degree of difficulty in performing the various transfection techniques can
vary significantly, and therefore the expertise and experience of the researcher is usually an
important factor to consider. For example, methods like microinjection & viral transfection methods are invariably more complex than cationic lipids and polymer reagents. Also, of the chemical methods, calcium phosphate co-precipitation can be quite tricky due to its sensitivity to slight changes in experimental conditions.
9. Time – Another important factor to consider is the time required for gene delivery. Viral
methods, while highly efficient, can be quite time consuming, typically taking anywhere from two weeks to one month to complete. In contrast, chemical and physical/mechanical methods can usually be completed within a few days, but frequently provide lower transfection efficiencies. Thus, it is not uncommon to have to weigh the benefits of faster results and less labor against the benefit of higher efficiency.
10 Cost – Finally, cost can be a major factor when deciding on the best gene delivery
method for a given application. The most expensive methods tend to be the
physical/mechanical methods, such as electroporation and gene guns, due to the specialized
equipment they require.Viral gene delivery is perhaps the second most expensive method, since it requires the vector reagents, extensive time and labor to complete lengthy vector preparation procedures, as well as proper disposal of viral waste materials.
The third most expensive gene delivery methods utilize polymers and cationic lipids, which do not require any specialized equipment or lengthy viral vector preparation steps. Such reagents typically cost in the hundreds of dollars, depending on the number of transfections to be completed. Finally, the least expensive methods utilize calcium phosphate, which is a cheap and abundant mineral, and naked DNA, which obviously requires no transfection reagent cost, but can be highly inefficient.
GENE TRANSFER TECHNIQUES
TRANSFORMATION
Transformation is the naturally occurring process of gene transfer which involves absorption of the genetic material by a cell through cell membrane causing the fusion of the foreign DNA with the native DNA resulting in the genetic expression of the received DNA. Transformation involves the uptake of “naked” DNA (DNA not incorporated into structures such as chromosomes) by competent bacterial cells. Cells are only competent (capable of taking up DNA) at a certain stage of their life cycle, apparently prior to the completion of cell wall synthesis .
Transformation is usually a natural method of gene transfer but as a result of technological advancement originated the artificial or induced transformation. Thus there are two types called as natural transformation and artificial or induced transformation. In natural transformation, the foreign DNA attaches itself to the host cell DNA receptor and with the help of the protein DNA translocase it enters the host cell. The presence of nucleases restricts the entry of two strands of the DNA, destroys a single strand thus allowing only one strand to enter the host cell. Any DNA that is not integrated into the chromosome will be degraded. This single stranded DNA mingles with the host genetic material successfully.
The artificial or induced method of transformation is done under laboratory condition which is either a chemical mediated gene transfer or done by electroporation.. Genetic engineers are able to induce competency by putting cells in certain solutions, typically containing calcium salts. At the entry site, endonucleases cut the DNA into fragments of 7,000-10,000 nucleotides, and the double-stranded DNA separates into single strands. The single-stranded DNA may recombine with the host’s chromosome once inside the cell. This recombination replaces the gene in the host with a variant – albeit homologous – gene.
Drawbacks:
DNA from a closely related genus may be acquired but, in general, DNA is not exchanged between distantly related microbes.
Not all bacteria can become competent.
CONJUGATION
Conjugation is a means of gene transfer in many species of bacteria. Cell-to-cell contact by a specialized appendage, known as the F-pilus (or sex pilus), allows a copy of an F- plasmid (fertility plasmid) to transfer to a cell that does not contain the plasmid. On rare occasions an F-plasmid may become integrated in the chromosome of its bacterial host, generating what is known as an Hfr (high frequency of recombination) cell. Such a cell can also direct the synthesis of a sex pilus. As the chromosome of the Hfr cell replicates it may begin to cross the pilus so that plasmid and chromosomal DNA transfers to the recipient cell. Such DNA may recombine with that of its new host, introducing new gene variants. Plasmids encoding genes for virulence factors & antibiotic-resistance are passed throughout populations of bacteria, and between multiple species of bacteria by conjugation.
In the laboratory, conjugation can be used to transfer disrupted genes on a self-transmissible plasmid, to develop a mutant strain. A gene of interest from a recipient E. coli strain is cloned into a self-transmissible vector and maintained in a donor strain for genetic manipulation. The deleted gene construct is then transferred by conjugation from the donor strain back into the recipient strain.
TRANSDUCTION
Transduction is another method for transferring genes from one bacterium to another; the transfer is mediated by bacteriophages. A bacteriophage infection starts when the virus injects its DNA into a bacterial cell. The bacteriophage DNA may then direct the synthesis of new viral components assembled in the bacterium. Bacteriophage DNA is replicated and then packaged within the phage particles. Early in the infective cycle the phage encodes an enzyme that degrades the DNA of the host cell. Some of these fragments of bacterial DNA are packaged within the bacteriophage particles, taking the place of phage DNA. As the number of phages increases it breaks open (lyse) the cell. When released from the infected cell, a phage that contains bacterial genes can continue to infect a new bacterial cell, transferring the bacterial genes.
Sometimes genes transferred in this manner become integrated into the genome of their new bacterial host by homologous recombination. Such transduced bacteria are not lysed because they do not contain adequate phage DNA for viral synthesis and these phages can enter an alternate life cycle called lysogeny. In this cycle, all the virus’s DNA becomes integrated into the genome of the host bacterium. The integrated phage, called a prophage, can confer new properties to the bacterium.
Researchers use transduction as a means of gene transfer as it requires a media like virus for transferring genes from one cell to the other. Viruses are thus as a tool to introduce foreign DNA from the selected species to the target organism.
There are two types of transduction called as generalized transduction in which any of the bacterial gene is transferred via the bacteriophage to the other bacteria and specialized transduction involves transfer of limited or selected set of genes.
Viral vectors for gene transfer:
The use of viruses as vectors for transduction, i.e. the introduction of genes into animal cells by exploiting the natural ability of the virus particle, within which the transgene is packaged, to adsorb to the surface of the cell and gain entry is being employed . Due to the efficiency with which viruses can deliver their nucleic acid into cells and the high levels of replication
and gene expression it is possible to achieve, viruses have been used as vectors not only for gene expression in cultured cells but also for gene transfer to living animals. Four classes of viral vector have been developed for use in human gene therapy and have reached phase 1 clinical trials. These are the retrovirus, adenovirus, herpesvirus and adenoassociated virus (AAV) vectors . Before introducing the individual vector systems,we discuss some general properties of viral transduction vectors. Transgenes may be incorporated into
viral vectors either by addition to the whole genome or by replacing one or more viral genes. This is generally achieved either by ligation (many viruses have been modified to incorporate unique restriction sites) or homologous recombination.
For many applications, it is favourable to use vectors from which all viral coding sequences have been deleted.These amplicons (also described as ‘gutless vectors’) contain just the cis-acting elements required for packaging and genome replication. The advantage of such vectors is their high capacity for foreign DNA and the fact that, since no viral gene products are made, the vector has no intrinsic cytotoxic effects. The choice of vector depends on the particular properties of the virus and the intended host, whether transient or stable expression is required and how much DNA needs to be packaged. For example, icosahedral viruses such as adenoviruses and retroviruses package their genomes into preformed capsids, whose volume defines the maximum amount of foreign DNA that can be accommodated. Conversely, rod-shaped viruses such as the baculoviruses form the capsid around the genome, so there are no such size constraints. There is no ideal virus for gene transfer ‘ each has its own advantages and disadvantages. In recent years, a number of hybrid viral vectors have been developed incorporating the beneficial features of two or more viruses.
Advantages:
1) viral vectors that especially efficient at transducing the intended cell type but not other cell types.
2) retroviruses are particularly proficient at delivering genes to dividing cells, such as cancer cells or immortalized cell lines
Limitations:
1) cannot deliver genes to terminally differentiated cells
2) viral vectors may be highly immunogenic
TRANSFECTION BY CHEMICAL METHODS
One of the methods of gene transfer where the genetic material is deliberately introduced into the animal cell in view of studying various functions of proteins and the gene is transfection. This mode of gene transfer involves creation of pores on the cell membrane enabling the cell to receive the foreign genetic material. When transformation is carried out in eukaryotic cells it is termed as transfection.
1) Calcium phosphate mediated DNA transfer
The process involves a mixture of isolated DNA (10-100ug) with solution of
calcium chloride and potassium phosphate. . When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface .Cells are then incubated with precipitated DNA either in solution or in tissue culture dish. A fraction of cells will take up the calcium phosphate DNA precipitate by a process not entirely understood but thought to be endocytosis.
Transfection efficiencies using calcium phosphate can be quite low, in the range of 1-2 % . It can
be increased if very high purity DNA is used and the precipitate allowed to form slowly.
Limitations
1. Frequency is very low.
2. Integrated genes undergo substantial modification.
3. Many cells do not like having the solid precipitate adhering to them
and the surface of their culture vessel.
4. Integration with host cell chromosome is random.
5, Due to above limitations transfection applied to somatic gene therapy
is limited.
Applications:
This technique is used for introducing DNA into mammalian cells. This process has been a preferred method of identifying many oncogenes.
2)DNA transfer by DAE-Dextran method:
DNA can be transferred with the help of DAE Dextran also. DAE-Dextran may be used in the
transfection medium in which DNA is present. This is a polycationic, high molecular weight substance and is convenient for transient assays. The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis.
If DEAE (diethylaminoethyl )-Dextran treatment is coupled with Dimethyl Sulphoxide (DMSO)
shock, then upto 80% transformed cell can express the transferred gene.
Advantages:
The advantage of this method is that, it is cheap, simple and can be used for transient cells
which cannot survive even short exposure of calcium phosphate.
The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency.
Limitations:
Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells.
Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection.
It does not appear to be efficient for the production of stable transfectants.
For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.
Transfer of DNA by polycation-DMSO:
As calcium phosphate method of DNA transfer is reproducible and efficient but there is narrow range of optimum conditions. DNA transfer by
Another polycationic chemical,the detergent Polybrene is carried out to increase the adsorption of DNA to the cell surface followed by a brief treatment with 25-30% DMSO to increase membrane permeability and enhance uptake of DNA. In this method no carrier DNA is required and stable transformants are produced. This method works with mouse fibroblast and chick embryo.
LIPOSOME MEDIATED GENE TRANSFER
Liposomes are spheres of lipids which can be used to transport molecules into the cells. These are artificial vesicles that can act as delivery agents for exogenous materials including transgenes. They are considered as spheres of lipid bilayers surrounding the molecule to be transported and promote transport after fusing with the cell membrane.
Liposomes for use as gene transfer vehicles are prepared by adding an appropriate mix of bilayer constituents to an aqueous solution of DNA molecules. In this aqueous environment, phospholipid hydrophilic heads associate with water while hydrophobic tails self-associate to exclude water from within the lipid bilayer. This self-organizing process creates discrete spheres of continuous lipid bilayer membrane enveloping a small quantity of DNA solution. The liposomes are then ready to be added to target cells. Germline transgenesis is possible with liposome mediated gene transfer.Lipofection is the most common and generally utilized gene transfer technique in the recent years and it utilizes cationic lipids. The combined DNA and cationic lipids act instantaneously to form structures called as lipoplexes that are more complex in structure than the simple liposomes. Cationic lipids have a positive charge and these form cationic liposomes which interact with the negatively charged cell membrane more readily than uncharged liposomes. This leads to a fusion between cationic liposome and the cell surface resulting in quick entry by endocytosis and the delivery of the DNA directly across the plasma membrane. Cationic liposomes can be produced from a number of cationic lipids that are commercially available and sold as an in vitro-transfecting agent, termed lipofectin.
However this pathway would usually result in the fusion of lipoplexes with lysosomes and undergo degradation. This problem is overcome by utilizing the neutral helper lipids, which are generally included along with the cationic lipid. This allows entrapped DNA to escape the endosomes, reach the nucleus and get access to the cell’s transcriptional machinery.The endosomal structure is destroyed by increasing the osmotic pressure created by the lipids’ buffering action within the endosomes and by the fusion of the lipid with the endosomal membrane. The ability of a lipid to destroy endosomes is one of the main characteristics of a transfection reagent.
Advantages
1. Simplicity.
2. Long term stability.
3. Low toxicity.
4. Protection of nucleic acid from degradation.
5. particularly proficient at delivering genes to dividing cells, such as cancer cells or immortalized cell lines
6. cationic lipids effectively transfect suspension cells.
Limitations:
1) Cannot deliver genes to terminally differentiated cells
ELECTROPORATION
A rapid and simple technique for introducing genes into a wide variety of microbial, plant and animal cells, including E. coli, is electroporation. Electroporation uses electrical pulse to produce transient pores in the plasma membrane thereby allowing macromolecules into the cells. These pores are known as electropores which allow the molecules, ions and water to pass from one side of the membrane to another. The electropores reseal spontaneously and the cell can recover. The pores can be recovered only if a suitable electric pulse is applied .The formation of electropores depends upon the cells that are used and the amplitude and duration of the electric pulse that is applied to them. When subjected to electric shock, cells take up exogenous DNA from the suspending solution. Once inside the cell, the DNA is integrated and the foreign gene will express. A proportion of these cells become stably transformed and can be selected if a suitable marker gene is carried on the transforming DNA. The most critical parameters are the intensity and duration of the electric pulse, and these must be determined empirically for different cell types. Electric currents can lead to dramatic heating of the cells that can results in cell death. Heating effects are minimized by using relatively high amplitude, a short duration pulse or by using two very short duration pulses. However, once optimal electroporation parameters have been established, the method is simple to carry out and highly reproducible.
Many different factors affect the efficiency of electroporation, including temperature,
various electric-field parameters (voltage, resistance and capacitance), topological form of the DNA,
and various host-cell factors (genetic background, growth conditions) and post-pulse treatment.
General applications of electroporation.
1. Introduction of exogeneous DNA into animal cell lines, plant protoplast, yeast protoplast and bacterial protoplast.
2. Electroporation can be used to increase efficiency of transformation or transfection of bacterial cells.
3. Wheat, rice, maize, tobacco have been stably transformed with frequency upto 1% by this method.
4. Genes encoding selectable marker may be used to introduce genes using electroporation.
5. To study the transient expression of molecular constructs.
6. Electroporation of early embryo may result in the production of transgenic animals.
7. Hepatocytes, epidermal cells, haematopoietic stem cells, fibroblast, mouse T and B lymphocytes can be transformed by this technique.
8. Naked DNA may be used for gene therapy by applying electroporation device on animal cells.
Advantages of electroporation.
1. Method is fast.
2. Less costly.
3. Applied for a number of cell types.
4. Simultaneously a large number of cell can be treated.
5. High percentage of stable transformants can be produced.
DRAWBACKs
1)Limited effective range of ~1 cm between the electrodes.
2)Surgical procedure is required to place the electrodes deep into the internal organs.
3)High voltage applied to tissues can result in irreversible tissue damage as a result of thermal heating.
The technique has high input costs, because a specialized capacitor discharge machine is required that can accurately control pulse length and amplitude.
4) Larger numbers of cells may be required than for other methods because, in many cases, the most efficient electroporation occurs when there is up to 50% cell death.
ULTRASONICATION
Sonication is the act of applying sound energy to agitate particles in a sample. Ultrasonic frequencies (>20 kHz) are usually used, leading to the process also being known as ultrasonication . Sonoporation, or cellular sonication, is the use of these ultrasonic frequencies for modifying the permeability of the cell plasma membrane. This technique is usually used in molecular biology and non-viral gene therapy in order to allow uptake of large molecules such as DNA into the cell, in a cell disruption process called transfection or transformation. Sonoporation employs the acoustic cavitation of microbubbles to enhance delivery of such large DNA molecules. Although the early results could achieve only 10- to 30-fold increases in transfection efficiency in vitro, technical refinements have lead to enhancements of up to several thousand fold in vitro ,sufficient to encourage ultrasonication mediated gene transfer in vivo.
Advantages:
1) It is non-invasive and well tolerated,
2)extraordinary safety record over a wide range of frequency and intensity
3)high levels of public acceptability and understanding.
there are highly sophisticated, flexible, cost-effective and readily available diagnostic and therapeutic systems that can achieve site-specific transfer of ultrasound energy almost anywhere in the body, except perhaps the lung.
4) The bioactivity of this technique is similar to, and in some cases found superior to, electroporation.
5) in vivo is relatively nontoxic.
Limitations:
1)Extended exposure to low-frequency (<MHz) ultrasound has been demonstrated to result in complete cellular death (rupturing) as well as microvascular hemorrhage and disruption of tissue structure, thus cellular viability must also be accounted for when employing this technique.
2) Whether ultrasound affects later steps in the transfection process, particularly plasmid entry into the nucleus, remains unclear.
Applications:
Sonoporation is under active study for the introduction of foreign genes in tissue culture cells, especially mammalian cells. Sonoporation is also being studied for use in targetedGene therapy in vivo, in a medical treatment scenario whereby a patient is given modified DNA, and an ultrasonic transducer might target this modified DNA into specific regions of the patient’s body.
MICROINJECTION
In microinjection DNA can be introduced into cells or protoplast with the help of very fine needles or
glass micropipettes having the diameter of 0.5 to 10 micrometer. The DNA may be directly delivered into the nucleus or in the cytoplasm. Some of the DNA injected may be taken up by the nucleus. The desired gene in the form of plasmid or alone is injected directly into the plant protoplast.
Injection into the nucleus is more efficient than that into the cytoplasm. Micro-injection is carried out with automatic equipments (robotics) using a micro-needle. Computerized control of holding pipette, needle, microscope stage and video technology has improved the efficiency of this technique.
Advantages of microinjection.
1. Frequency of stable integration of DNA is far better as compare to other methods.
2. Method is effective in transforming primary cells as well as cells in established cultures.
3. The DNA injected in this process is subjected to less extensive modifications.
4. Mere precise integration of recombinant gene in limited copy number can be obtained.
Limitations
1. Costly.
2. Skilled personal required.
3.Has not been successful for many plant cells .
4. Embryonic cells preferred for manipulation.
5. Knowledge of mating timing, oocyte recovery is essential.
6. Method is useful for protoplasts and not for the walled cells.
7. Process causes random integration.
8. Rearrangement or deletion of host DNA adjacent to site of integration are common.
9. The technique is laborious, technically difficult, and limited to the number of cells actually injected.
Applications of microinjection.
1. Process is applicable for plant cell as well as animal cell but more common for animal cells.
2. Technique is ideally useful for producing transgenic animal quickly.
3. Procedure is important for gene transfer to embryonic cells.
4. Applied to inject DNA into plant nuclei.
5. Method has been successfully used with cells and protoplast of
tobacco, alfalfa etc.
Fig: microinjection in mouse egg.
BIOLISTICS
(MICROPROJECTILE/GENE GUN)
Biolistics or particle bombardment is a physical method that uses accelerated microprojectiles to
deliver DNA or other molecules into intact tissues and cells. Biolistics transformation is relatively new and novel method amongst the physical methods for artificial transfer of exogenous DNA. The nucleic acid is delivered through membrane penetration at a high velocity, usually connected to microprojectiles like microscopic particles of tungsten or gold coated into cells using an electrostatic pulse, air pressure, or gunpowder percussion. As the particles pass through the cell, the DNA becomes free to integrate into the plant-cell genome.
The gene gun is a device that literally fires DNA into target cells . The DNA to be transformed
into the cells is coated onto microscopic beads made of either gold or tungsten. Beads are carefully
coated with DNA. The coated beads are then attached to the end of the plastic bullet and loaded into
the firing chamber of the gene gun. An explosive force fires the bullet down the barrel of the gun
towards the target cells that lie just beyond the end of the barrel. When the bullet reaches the end of
the barrel it is caught and stopped, but the DNA coated beads continue on toward the target cells.
Some of the beads pass through the cell wall into the cytoplasm of the target cells. Here the bead and
the DNA dissociate and the cells become transformed. Once inside the target cells, the DNA is
solubilised and may be expressed.
Applications
1. Biolistics technique has been used successfully to transform soyabean,
cotton, spruce, sugarcane, papaya, corn, sunflower, rice, maize, wheat,
tobacco etc.
2. Genomes of subcellular organelles have been accessible to genetic
manipulation by biolistic method.
3. Mitochondria of plants and chloroplast of chlamydomonas have been
transformed.
4. Method can be applied to filamentous fungi and yeast (mitochondria).
5. The particle gun has also been used with pollen, early stage
embryoids, meristems somatic embryos, plant cells, root section, seeds and pollen.
6. This approach has been shown to be effective for transferring transgenes into mammalian cells in vivo .
Advantages
1. Requirement of protoplast can be avoided. Unlike electroporation and microinjection, this technique does not require protoplasts or even single-cell isolations and is applicable to even intact plant tissue
2. Walled intact cells can be penetrated.
3. Manipulation of genome of subcellular organelles can be achieved.
4. This is also a highly mechanized or robotic mediated technique in which the
speed of the micro-projectile particles is controlled by foolproof mechanisms
5) It is also gaining in use as a method for transferring DNA construct into whole animals.
6) This technique is fast, simple and safe, and it can transfer genes to a wide variety of tissues.
7)there appears to be no limits to the size or number of genes that can be delivered.
Limitations
1. Integration is random.
2. Requirement of equipments.
3.It can be a challenge to obtain a sufficient number of cells modified by this method to see a biologically significant effect.
MICROLASER
A new technique is presented to incorporate exogeneous gene materials (DNA) into cells with a microbeam irradiation from an uv pulsed laser. A frequency-multiplied Laser, 355 m wavelength, 5 ns pulse duration, punches a self-healing hole of submicrometer aperture in cell membrane under selected irradiation conditions. At the site of the beam impact, due probably to local temperature changes, the cell membrane modifies its permeability. As a consequence of the hit, circular areas, whose radius may be apparently regulated by changing the irradiation time and/or the radiation intensity (energy), appear on the wall, last for a short time and fade spontaneously . It takes a fraction of a second for the aperture to close, long enough to allow the foreign DNA, contained in the medium, to slip into the cell.
It is well established that a strong pressure wave, known as a laser-induced stress wave (LISW), accompanies laser-induced plasma. We have extended their method to deliver macromolecules, such as genes. Plasmid DNA (circular DNA residing in bacterial cytoplasm) is used as a vector of the gene of interest. We inject the plasmid into target tissue, on which a laser target is placed, and subject the target to irradiation with a high-intensity, nanosecond laser pulse to induce plasma and hence an LISW. By placing optically transparent material on the target, the plasma is confined, resulting in an increase in the LISW’s impulse. Interaction of tissue cells with the LISW allows plasmid to enter the cytoplasm
Advantages
1) it only takes advantage of the presence of phenol-red, a normal cell medium component, with no need of addition of extraneous substances;
(2) it is a very mild treatment virtually suitable for any cell type and
(3) it allows transfection of selected cells even in the presence of cells of different type (providing that they are morphologically distinguishable).
4) . it is rapidly replacing virus mediated techniques as they have serious side effects caused by immune response and limited targeting characteristics rendering them inappropriate for clinical application
5) The method offers a clear advantage over existing methods: increases the success rate of DNA transfection as well as the efficiency of cell modification by orders of magnitude.
6) . It enables minimally invasive tissue interaction.
CONCLUSION
Thus there are a number of ways by which the genes can be introduced into the cells. With the advent of molecular tools and technologies it is now comparatively easy to introduce gene into cells without losing its integrity and biological activity. Moreover the recent development in molecular biology has made the transfer of gene with great accuracy to the target cell. The transfer of gene through different gene transfer technologies has cured a number of diseases. Research is on progress to cure those diseases which cannot be cured by using drugs. Moreover the treatment of diseases by gene transfer provides better result for a prolong period of time. It is the need of hour to discover new and cheap method of gene transfer technologies so to make the treatment of the diseases a little easier and affordable. Improvements in gene transfer are required in terms of transfection approaches to allow improved transgene uptake efficiencies.
REFERENCES
1)Society of Applied Sciences.
Gene Transfer Technologies and their Applications: K.H.KHAN
Medical Biotechnology Division, School of Biosciences and Technology,
VIT University, Vellore-632014, Tamil Nadu, India.
2)Principles of gene manipulation: R.W.Old ,S.B. Primrose and R.M.Twymann ‘ sixth edition,chapter 10 and 11.
3) BIOTECHNOLOGY 2020-From the Transparent Cell to
the Custom-Designed Process–Prof. Gerhard Kreysa Dr.-Ing. E.h. Dr.h.c. & Dr. R??diger Marquardt
4) INTRODUCTION TOBIOTECHNOLOGY AND GENETIC ENGINEERING
-A.J. NAIR, PH.D.
5)From genes to genomes- concepts and applications of DNA technology–Jeremy W Dale, Malcom von Schantz
6) http://www.nature.com
7)www.learner.org
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