Microprojectile Bombardment (Gene Gun Method): Principle, Steps, Applications, Advantages & Disadvantages

Table of Contents

• Introduction to Microprojectile Bombardment
• Principle of Microprojectile Bombardment
• Microprojectile Bombardment Instruments
• Steps in Microprojectile Bombardment
• Applications of Microprojectile Bombardment
• Advantages of Microprojectile Bombardment
• Disadvantages of Microprojectile Bombardment
• References

Introduction to Microprojectile Bombardment

• Microprojectile Bombardment is a physical method of gene transfer that introduces foreign genetic material directly into cells or tissues by using high-velocity particles.
• The technique is also referred to by several alternative names, including Particle Bombardment, Particle Gun, Ballistics, and Particle Acceleration.
• The concept of Microprojectile Bombardment was developed by John Sanford and Ed Wolf.
• This method was originally designed for transforming plant cells but was later adapted for use in mammalian cells as well.

Principle of Microprojectile Bombardment

• The fundamental principle of microprojectile bombardment is the direct delivery of genetic material into target cells by bombarding them with microcarrier particles at very high velocity.
• Microcarrier particles are typically composed of inert metals such as gold or tungsten and are coated with the desired genetic material.
• These coated particles are propelled into the target cells using a high-velocity stream, which is usually generated either by an electric discharge or a helium pulse.
• The high-velocity propulsion drives the microcarrier particles into the target cells, enabling them to penetrate the cell membrane and reach the cytoplasm.
• Once inside the cells, the genetic material detaches from the microcarrier particles.
• The released genetic material is then available for utilization by the cellular machinery, leading to gene expression.

Microprojectile Bombardment Instruments

• Various types of gene guns have been developed for microprojectile bombardment.
• The first-generation microprojectile bombardment devices initially used gunpowder to propel microcarrier particles coated with the desired genetic material.
• Gunpowder-based systems were later replaced by high-pressure helium for particle acceleration, which significantly improved transformation efficiency.
• Technological advancements have led to improved gene guns that employ different propulsion forces, including electrostatic, pneumatic, and compressed gas systems.
• Two widely used devices for microprojectile bombardment are the PDS-1000/He and the Helios gene gun.
• The PDS-1000/He utilizes helium gas to accelerate microscopic gold or tungsten particles coated with genetic material toward the target tissue.
• The Helios gene gun is a handheld device designed for transforming larger target tissues.
• Both the PDS-1000/He and the Helios gene gun use pressurized helium to propel microcarrier particles coated with genetic material.

Steps in Microprojectile Bombardment

• The microprojectile bombardment process begins with preparing microcarrier particles, which are typically made of gold or tungsten, for coating with the genetic material of interest.
• The desired genetic material is then coated onto the surface of these microcarrier particles.
• After coating, the microcarrier particles are loaded into the gene gun device.
• The gene gun propels the particles at high velocity using a strong pulse of pressurized helium.
• When the pressure inside the gene gun reaches a critical level, the rupture disk bursts, creating a powerful wave of gas.
• This gas wave pushes the macrocarrier carrying the microcarrier particles toward the target cells.
• The macrocarrier strikes the stopping screen, which prevents the macrocarrier itself from passing but allows the coated microcarrier particles to continue toward the target cells.
• The target cells are placed in the main chamber of the gene gun under a vacuum, typically on a petri dish or culture plate.
• The high-velocity microcarrier particles penetrate the cell membrane of the target cells and enter the cytoplasm.
• Once inside the cells, the genetic material separates from the microcarrier particles.
• The released genetic material is then available for utilization by the cell’s machinery, leading to gene expression.

Applications of Microprojectile Bombardment

• Some major applications of microprojectile bombardment are:
• Microprojectile bombardment is widely used for introducing foreign genes into plant cells and producing genetically modified plants with improved traits such as disease resistance and higher yield.
• Microprojectile bombardment has also been used to generate transgenic animals with specific desired traits.
• This method of gene delivery allows the study of gene function and expression patterns in different tissues.
• This method also has applications in gene therapy for delivering therapeutic genes directly into target tissues to treat genetic disorders, cancer, and other diseases.
• Microprojectile bombardment can also be used to develop DNA vaccines by delivering DNA-encoding antigens directly into cells.
• It can also be used to deliver fluorescent dyes into cells and tissues to study cellular signaling processes.

Plant transformation via particle bombardment involves a series of steps: (1) the process begins with the mother plant, (2) from which a leaf is selected, (3) and the leaf cells are isolated. (4a) The cell walls of these isolated cells are removed, and (4b) protoplasts are obtained. (5) Gene transfer is then carried out using a particle bombardment device. (6) Following this, callus induction takes place, (7) which is followed by regeneration of transformed tissues. (8) Rooting occurs as the tissues develop further, (9) eventually resulting in the formation of a young plantlet. (10) Finally, molecular and histo-chemical analyses are performed to confirm successful transformation.

Advantages of Microprojectile Bombardment

• Microprojectile bombardment is a fast and relatively simple method for delivering desired genetic material into cells.
• It allows the delivery of large nucleic acid fragments.
• The method is not limited by host specificity or species, making it widely applicable.
• It is a safer approach for genetic transformation because it does not require harmful viruses or toxic chemicals as gene delivery vehicles.
• Unlike other methods that require removal of the cell wall, microprojectile bombardment can penetrate intact cell walls, simplifying the process and enabling transformation of a broader range of cells.

Disadvantages of Microprojectile Bombardment

• Microprojectile bombardment requires specialized equipment, and the initial investment for the necessary devices and materials can be expensive.
• Target cells may undergo physical damage from the high-velocity particles, which can reduce cell viability.
• The effectiveness of the gene gun method tends to decrease when applied on a larger scale.
• Introduced DNA may integrate randomly into the host genome, potentially leading to unpredictable patterns of gene expression.

References

Baltes, N. J., Gil-Humanes, J., & Voytas, D. F. (2017). Genome engineering and agriculture: Opportunities and challenges. In Gene Editing in Plants (pp. 1–26). https://doi.org/10.1016/bs.pmbts.2017.03.011

Carter, M., & Shieh, J. (2015). Gene delivery strategies. In Guide to Research Techniques in Neuroscience (pp. 239–252). https://doi.org/10.1016/b978-0-12-800511-8.00011-3

Do Minh, A., Sharon, D., Chahal, P., & Kamen, A. A. (2019). Cell transfection. In Comprehensive Biotechnology (pp. 383–390). https://doi.org/10.1016/b978-0-444-64046-8.00023-9

Biology Reader. (n.d.). Microprojectile bombardment. Retrieved from https://biologyreader.com/microprojectile-bombardment.html

Jinturkar, K. A., Rathi, M. N., & Misra, A. (2011). Gene delivery using physical methods. In Challenges in Delivery of Therapeutic Genomics and Proteomics (pp. 83–126). https://doi.org/10.1016/b978-0-12-384964-9.00003-7

Matsumoto, T. K., & Gonsalves, D. (2012). Biolistic and other non-Agrobacterium technologies of plant transformation. In Plant Biotechnology and Agriculture (pp. 117–129). https://doi.org/10.1016/b978-0-12-381466-1.00008-0

Ozyigit, I. I., & Yucebilgili Kurtoglu, K. (2020). Particle bombardment technology and its applications in plants. Molecular Biology Reports. https://doi.org/10.1007/s11033-020-06001-5