Microparticle Bombardment – BIOLISTICS (Gene Gun)
Microparticle or microprojectile bombardment is a direct method of transferring the gene of interest to target cells. This gene of interest is an exogenous genetic material that is desired to be incorporated into the targeted cell. The genetic material could be DNA, RNA, or proteins. Microprojectile bombardment is also called gene gun delivery or particle acceleration. It is a very efficient method of physical transfer of gene transfer in plants. It’s predominantly used for gene transfer in plants. Microparticle bombardment employs high-speed particle bombardment for gene transfer. However, it is also used for the transfer of genes in mammalian cells and microorganisms. The microprojectile bombardment method was also named biolistics or gene gun.
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History Behind the Invention of BIOLISTICS
The earlier used method of gene transfer that is the agrobacterium-mediated genetic transformation system worked well for dicotyledonous plants but has low efficiency for monocots. John Stanford in 1984, invented this technique for the introduction of DNA into cells by physical means to avoid the host-range restrictions of Agrobacterium. The microprojectile gun has been proven to be a successful alternative technique for creating transgenic organisms in prokaryotes, mammalian, and plant species.
What’s the methodology of Microparticle bombardment in BIOLISTICS?
Biolistics or microprojectile is a method where the cells are physically impregnated with nucleic acids or other biological molecules. In this particle delivery system, the cells to be transformed are bombarded with heavy metal particles coated with DNA/RNA. The DNA construct is first mixed with particles of a heavy metal, usually tungsten or gold(0.6 – 1 mm in size). When these fine particles stick to the negatively charged DNA and are loaded on one side of a plastic bullet.
- Firstly, before coating DNA is precipitated with calcium chloride, spermidine, and polyethylene glycol.
- These coated microparticles are then loaded onto the macro carrier and accelerated to high speed by using pressurized helium gas.
- The predominant targets of the gene gun are plant cell suspensions, callus cultures of undifferentiated plant cells, or a group of immature embryos growing on a gel medium in a Petri dish.
- Modern applications of biolistics are in the bombardment of animal cells as well.
- Once the target is aimed, the microprojectile penetrates the cell wall and cell membrane of the cell. Then the coated DNA is released from its surface such that the target cell’s genome gets incorporated.
Applications of Microparticle Bombardment or BIOLISTICS
Research has shown that this method of gene transfer is being proven a successful alternative to existing techniques for creating transgenic organisms. Transgenic organisms also called Genetically Modified Organisms (GMOs) developed by this method could be both plants and animals.
- There has been tremendous success in transforming plant species. It is due to its rapid assessment of the transient expression of genetic constructs introduced into cells of intact tissues. After the plant cell genome gets impregnated by the DNA-coated gold particles, the DNA is used as a template strand for transcription by the cell. This is called transient expression.
- The capability of biolistics to optimize the delivery of DNA into the cells is done on strict parameters. These include the target tissue, behavior in tissue culture, and available marker genes. If the delivered DNA construct contains a selectable marker, then stably transformed cells can be selected and cultured accordingly using tissue culture methods.
- For example, transformed cells in the culture with a DNA construct containing a gene that confers resistance to any particular antibiotic or herbicide, are selected including that antibiotic or herbicide in the tissue culture media.
- The selected transformed cells are allowed to divide and differentiate into the organized, specialized, tissue cells of an entire plant. It happens when the transformed cells are treated with a series of plant growth hormones, such as auxins and gibberellins. This capability in plants for total regeneration is called totipotency.
- Thus, the desired gene can be introduced into callus cultures or even tissues of whole seedlings.
One such successful attempt of the particle bombardment technique was when Bt-toxin genes were incorporated in MAIZE for a producing transgenic plant.
In the case of animals, the particle bombardment method is generally used for delivering DNA vaccines. It has been able to transfect DNA plasmid to animal cells that code for fluorescent proteins. Apart from making it a protein-churning factory, they are also used to deliver a vital variety of dyes to cells.
Advantages Of Microparticle Bombardment or BIOLISTICS
- In contrast to the Agrobacterium-mediated transformation method, which is quite difficult to target the vector, it has proven to be a versatile method of genetic modification.
- For many species which are not amenable to vector-mediated transformation, gene gun comes into the picture. It is species independent and can be used successfully for a wide range of organisms.
- Transformation of organelle DNA (mitochondria and chloroplasts) has also been made possible by this method. Plastid transformation is one such achievement of particle bombardment.
- Biolistics has been used in gene transfer to intact plant tissues
- Multiple genes can be introduced in a single plant using it.
Limitations Of Microparticle Bombardment or BIOLISTICS
- Due to that lack of proper construct, sometimes the DNA may be transformed into whatever genomes are present in the cell, be it nuclear, mitochondrial, or plastid genomes.
- Limited regeneration capacity of tissue being bombarded.
- Highly specialized and expensive equipment is required.
- The tissues of the target might get damaged due to high bombardment velocity.
- There can be the insertion of multiple copies of the gene.
- At times such gene transfer techniques give rise to undesirable genetic transformations and thus unwanted transgenic organisms.
With the growing advancements in the field of biotechnology, this method of gene delivery gains the limelight especially when past success records are considered. Though the apparatus and setup still demand high installation charges, microparticle bombardment is more efficient than the previously used Agrobacterium-mediated transformation. This technique is able to transform all types of cells, different species, and even different organelles, that is it is not restricted only to the nucleus. Presently gold is highly favoured for bombardment. This is due to its lower cytotoxicity in comparison to tungsten projectile carriers.