Physical method of transformation- Microprojectile gun Bombardment, Electroporation and Microinjection

1. PHYSICAL METHOD OF TRANSFORMATION
Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to Bengaluru North University,
K. Narayanapura, Kothanur (PO)
Bengaluru 560077

2. PHYSICAL METHOD OF TRANSFORMATION:
Due to amphipathic nature of the phospholipid bilayer of the plasma membrane, polar
molecules such as DNA and protein are unable to freely pass through the membrane.
Various physical or mechanical methods are used in gene transfer in overcoming this
barrier.
They are:
1. Electroporation
2. Microinjection
3. Particle Bombardment
4. Sonoporation
5. Laser induced
6. Bead Transfection

3. I). ELECTROPORATION:
This method was first demonstrated by Wong and Neumann in 1982 to study gene transfer
in mouse cells.
Electroporation was introduced in the 1960s and comprises the application of controlled
electric fields to facilitate cell permeabilization. The success of in vitro delivery by
electroporation has led to the development of in vivo applications. The first in vitro and in
vivo attempts to use electroporation in gene transfer were demonstrated in 1982 and 1991,
respectively.
Electroporation is a physical transfection method that uses an electrical pulse that
temporarily disturbs the phospholipid bilayer and create temporary pores in cell
membranes through which substances like nucleic acids can pass into cells.
It is now a widely used method for the introduction of transgene either stably or transiently
into bacterial, fungal, plant and animal cells.
It is a highly efficient strategy for the introduction of foreign nucleic acids into many cell
types, including bacteria and mammalian cells.

4. Principle:
Electroporation is based on a simple process.
 Host cells and selected molecules are suspended in a conductive solution, and an
electrical circuit is closed around the mixture.
 Conditions: varying conditions of Voltage (V), resistance(Ω) and capacitor (µF) 25
 An electrical pulse at an optimized voltage and only lasting a few microseconds to a
millisecond is discharged through the cell suspension.
This disturbs the phospholipid bilayer of the membrane and results in the formation of
temporary pores.
The electric potential across the cell membrane simultaneously rises to allow charged
molecules like DNA to be driven across the membrane through the pores in a manner similar
to electrophoresis.

5. Electroporation buffers
Choice of electroporation buffer depends on the cells being used in the experiment.
The following buffers (stored at 4°C) can be used:
1. Phosphate Buffered Saline (PBS) without Ca++ or Mg++
2. HEPES-buffered saline
3. Tissue culture medium without FCS
4. Phosphate-buffered sucrose: 272 mM sucrose/7 mM K2HPO4 (adjusted to pH 7.4
with phosphoric acid)/1 mM MgCl2
5. 0.4 M mannitol in PBS
6. 5 mM CaCl2 in PBS
7. 0.5 M sucrose
Electroporation Cuvettes: For electroporation of microorganisms, 0.1 and 0.2 cm gap
cuvettes are most often used.
Electroporation of E. coli is generally carried out:
•at a voltage of 1.8 kV when electroporating cells in 0.1 cm cuvettes and
•at a voltage of 2.5 kV when electroporating cells in 0.2 cm cuvettes.

6. Procedure:
1. A population of target cells is taken and resuspended the cells with the electroporation
buffer. The electroporation buffer protects cell suspension from mechanical damage.
2. Along with it, target DNA or plasmid DNA taken and mixed in the cuvette. Then it is
placed in an electroporator.
3. The electroporation cuvette with two aluminium electrodes, carrying the target cell
suspension mixed with plasmid or target gene, is fitted in the electroporation machine
or device known as electroporator.
4. When the device is connected with the power source, set with voltage (1200-2500V),
resistance (Ohm-Ω) and constant capacitor (25 or 50µF) and when it is switched on,
the circuit completes, and the pulsed is generated.

7. 5. An electrical pulse is passed through the cell suspension. This will create
temporary/transient pores in the cell plasma membrane. Charged molecules like DNA can
pass through pores into the cytoplasm of the cell, under the influence of current.
6. Note: The entire process completes within a second (in a microsecond or millisecond). The
success rate of the entire process depends on two variables; pulse length and field
strength.
7. Immediately, the cells are suspended into nutrient media, to revive the cells for 1-2 hr at
37°C and the viability/transformed cells are checked by plating the electroporated cells
onto the media containing respective antibiotic or other markers.

8. Applications
Electroporation is widely used in many areas of molecular biology and in medical field.
Some applications of electroporation include:
DNA transfection or transformation
Electroporation is mainly used in DNA transfection/transformation which involves
introduction of foreign DNA into the host cell (animal, bacterial or plant cell).
Direct transfer of plasmids between cells
It involves the incubation of bacterial cells containing a plasmid with another strain lacking
plasmids but containing some other desirable features. The voltage of electroporation
creates pores, allowing the transfer of plasmids from one cell to another. This type of transfer
may also be performed between species. As a result, a large number of plasmids may be
grown in rapidly dividing bacterial colonies and transferred to yeast cells by electroporation.
Gene transfer to a wide range of tissues
Electroporation can be performed in vivo for more efficient gene transfer in a wide range of
tissues like skin, muscle, lung, kidney, liver, artery, brain, cornea etc. It avoids the vector-
specific immune-responses that are achieved with recombinant viral vectors and thus are
promising in clinical applications.

9. Advantages
1. It is highly versatile and effective for nearly all cell types and species.
2. It is highly efficient method as majority of cells take in the target DNA molecule.
3. It can be performed at a small scale and only a small amount of DNA is required as
compared to other methods.

10. II). MICROINJECTION
DNA microinjection was first proposed by Dr. Marshall A. Barber in the early of nineteenth
century.
1. This method is widely used for gene transfection in mammals.
2. This method has been successfully applied to genetically modified animals including
mice, fish, rats, rabbits and many large domestic animals such as cattle, sheep and pigs.
3. It involves delivery of foreign DNA into a living cell (e.g. a cell, egg, oocyte, embryos of
animals) through a fine glass micropipette. The introduced DNA may lead to the over or
under expression of certain genes.
4. It is used to identify the characteristic function of dominant genes.

11. Procedure:
1. The delivery of foreign DNA is done under a powerful microscope using a glass
micropipette needles with very fine tip (0.1 to 0.5 μm) to directly inject foreign gene
fragments into pronuclear embryos or cultured cells.
2. Cells to be microinjected are placed in a container. A holding pipette is placed in the
field of view of the microscope that sucks and holds a target cell at the tip.
3. The tip of micropipette is injected through the membrane of the cell to deliver the
contents of the needle into the cytoplasm and then the empty needle is taken out.
Delivery of DNA into a cell through microinjection

12. Advantages
1. • No requirement of a marker gene.
2. • Introduction of the target gene directly into a single cell.
3. • Easy identification of transformed cells upon injection of dye along with the DNA.
4. • No requirement of selection of the transformed cells using antibiotic resistance or
herbicide resistance markers.
5. • It can be used for creating transgenic organisms, particularly mammals.
Disadvantages
1. Injection can cause damage that affects embryonic survival and can result in quite
high mortalities.
2. Only one cell is targeted per injection.

13. III.) PARTICLE BOMBARDMENT METHOD
• It was invented by Prof. John C Sanford, Ed Wolf and Nelson Allen at Cornell University
(USA), and Ted Klein of DuPont, between 1983 and 1986 developed the original
bombardment concept and coined the term “biolistics” (short for “biological ballistics”) for
both the process and the device.
• The original target was onions (chosen for their large cell size) and it was used to deliver
particles coated with a marker gene. Genetic transformation was then proven when the
onion tissue expressed the gene.
• Also termed as particle bombardment, particle gun, shot gun, micro projectile
bombardment and particle acceleration.
• It employs high-velocity microprojectiles to deliver substances into cells and tissues.
Macrocarrier

14. Apparatus
1. The biolistic gun involves the principle of the passage of helium gas through the cylinder
with arrange of velocities required for optimal transformation of various cell types.
2. It consists of a bombardment chamber which is connected to an outlet for vacuum
creation.
3. The bombardment chamber consists of a plastic rupture disk below which macrocarrier is
loaded with micro carriers.
4. These microcarriers consist of gold or tungsten micro pellets coated with DNA for
transformation.
B
Macrocarrier

15. 5. The apparatus is placed in Laminar flow to maintain sterile conditions.
6. The target cells/tissue is placed in the apparatus and a stopping screen is placed
between the target cells and micro carrier assembly.
7. The passage of high pressure helium ruptures the plastic rupture disk propelling the
macrocarrier and microcarriers.
Working system of particle bombardment gun
8. The stopping screen prevents the passage of macro projectiles but allows the DNA
coated micro pellets to pass through it thereby, delivering DNA into the target cells.
Macrocarrier

16. Procedure:
Step 1: The gene gun apparatus is ready to fire. Helium fills the chamber and pressure
builds against the rupture disk.
Step 2: The DNA to be transformed into the cells is coated onto microscopic beads
made of either tungsten or gold. The coated beads are then attached to the end of the
plastic bullet (macrocarrier) and loaded into the firing chamber of the gene gun.
Step 3: The pressure eventually reaches the point where the rupture disk breaks, and
the resulting burst of helium propels macrocarrier and hits the DNA/gold-coated
microcarrier into the stopping screen.
Step 4: when the macrocarrier hits the stopping screen, the DNA-coated gold particles
are propelled through the screen and into the target cells.
Macrocarrier
The stopping screen prevents the passage of macro projectiles but
allows the DNA coated micro pellets to pass through it thereby,
delivering DNA into the target cells

17. Uses
1. • This method is commonly employed for genetic transformation of plants and many
organisms. This technique has been used successfully to transform soyabean, cotton,
spruse, sugarcane, papaya, sunflower, rice, maize, wheat and tobacco.
2. • The particle gun has also been used with pollen, early stage embroyoids, meristems and
somatic embryos.
3. • This method is applicable for the plants having less regeneration capacity and those
which fail to show sufficient response to Agrobacterium- mediated gene transfer in rice,
corn, wheat, chickpea, sorghum and pigeon-pea.
4. Gene guns have also been used to deliver DNA vaccines.
5. The delivery of plasmids into rat neurons through the use of a gene gun, specifically DRG
neurons, is also used as a pharmacological precursor in studying the effects of
neurodegenerative diseases such as Alzheimer's disease.
6. The gene gun has become a common tool for labeling subsets of cells in cultured tissue. In
addition to being able to transfect cells with DNA plasmids coding for fluorescent proteins,
the gene gun can be adapted to deliver a wide variety of vital dyes to cells.
7. Gene gun bombardment has also been used to transform Caenorhabditis elegans, as an
alternative to microinjection.

18. Advantages
• Simple and convenient method involving coating DNA or RNA on to gold microcarrier,
loading sample cartridges, pointing the nozzle and firing the device.
• No need to obtain protoplast as the intact cell wall can be penetrated.
• Manipulation of genome of sub-cellular organelles can be done.
• Eliminates the use of potentially harmful viruses or toxic chemical treatment as gene
delivery vehicle.
• This device offers to place DNA or RNA exactly where it is needed into any organism.
• Technical simplicity and Relatively high transformation efficiency.
Disadvantages
• The transformation efficiency may be lower than Agrobacterium- mediated
transformation.
• Specialized equipment is needed. Moreover the device and consumables are costly.
• Associated cell damage can occur.
• The target tissue should have regeneration capacity.
• Random integration is also a concern.
• Chances of multiple copy insertions could cause gene silencing.