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ISSN 2320-6020

IJBSTR REVIEW PAPER (RVP-2) VOL 1 [ISSUE 2] FEBRUARY 2013

Biolistic Mediated Gene Transfer


N.W. Deshmukh* and Hardeep
ABSTRACT: The ability to deliver foreign DNA directly into regenerable cells, tissues, or organs
appears to provide the best method, at present, to achieve truly genotype-independent transformation in many
agronomic crops, bypassing Agrobacterium host-specificity and tissue culture-related regeneration difficulties.
Microprojectile bombardment employs high-velocity metal particles to deliver biologically active DNA into
plant cells. In addition to the transformation of recalcitrant agronomic crops, woody species have been also
engineered using this technology.
Keywords : Gene gun, Biolistic Method, Transformation, Agrobacterium, Microprojectile
INTRODUCTION
article bombardment is a physical method
of cell transformation in which high
density, sub-cellular sized particles are
accelerated to high velocity to carry DNA into
cells. The technique was first described as a
method of gene transfer into plants (Klein et
al., 1987, 1988; McCabe et al., 1988) and
subsequently shown to be applicable to
mammalian experimental systems (Zelenin et
al., 1989; Yang et al., 1990; Williams et al.,
1991). Because it does not depend on specific
ligand-receptors and/or the biochemical
features of structural components typically
present on cell surfaces, particle-mediated gene
transfer can be readily applied to a variety of
biological systems. Consequently, this
procedure can be used to transform such
diverse targets as bacteria (Shark et al., 1991;
Smith et al., 1992), fungi (Armaleo et al.,
1990), and intracellular organelles (Johnston et
al., 1988; Boynton et al., 1988).

Since it is a physical method of gene delivery,


particle bombardment also overcomes physical
barriers to effective gene transfer, such as the
stratum corneum of the epidermis and the cell
wall of plants. Particle bombardment is a
convenient method for transforming intact
cells in culture since minimal pre- or postbombardment manipulation is necessary. In
addition, this technique is much easier and
faster to perform than the tedious task of
microinjection. Both transient and stable
expression are possible with particle
bombardment. In addition to DNA, RNA may
also be transferred to cells by particle
bombardment (Qiu, et al., 1996).

Author: N. W. Deshmukh, LPU, Jalandhar, Punjab


(India), Email- Hnikten4you@gmail.com
Co-Author- Hardeep, Chiheru, Kapurthala, Ponjab
(India), Email- harryhardeep503@gmail.com

Fig: 1.1: Transfection through gene gun

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The Gene Gun is a portable particle


Advantages of particle bombardment for in
bombardment device. The range of its use is
vitro and in vivo gene transfer.
limited by its requirement for a supply of
Easy to use, rapid, versatile gene delivery pressurized helium and the 6 foot length of
pressurized helium hose. When using the Gene
system
Gun, only a small area is needed for setting
Independent of target cell type
Useful for both transient and stable down the gun during an experiment, for
loading the cartridges into the cartridge holders
expression
and exchanging cartridge holders during
Requires only small amounts of DNA
experiments. In addition, a clean and dry area
No carrier DNA is needed
is needed for working with the tissue samples.
Requires only small numbers of cells
Preparation of the gold/DNA tubes used in the
May obtain high levels of co-transformation
Gene Gun requires an area approximately 1 m2
Large DNA fragments may be transferred
Direct intracellular delivery to many cells in for the Tubing Prep Station, for manipulating
the tubing, precipitating the DNA onto the
the target area
Applicable to both in vitro and in vivo gold, and processing the tubing into cartridges.
Additionally, the Tubing Prep Station requires
transformation
No extraneous genes or proteins are delivered an electrical outlet and a tank of pressurized
nitrogen for evaporating the ethanol from the
DNA-coated gold particles from the inner
Operating Principle
surface of the tubing.
It uses micron-size carrier particles
made of a heavy metal (tungsten or gold) that Helium Supply
are coated with plasmids, RNA, or dye,
Only helium gas is to be used with the
accelerated to a high speed using a pneumatic
gun (Gene Gun) and launched into a biological Gene Gun. The low atomic weight of helium
target (cultured cells, tissue, plant, small results in maximum gas expansion when the
animal). A small bead penetrates cells high pressure helium is released through the
without damaging them, gets stuck in a cell in valve opening and enters the cartridge at
pressure. Thus,
sufficient
an internal layer of the targeted tissue, and atmospheric
releases into the cells the chemicals that it acceleration of the DNA-coated microcarriers
carries. The technique of biolistic delivery has is generated for penetration of the target cell
been used for transfection (beads coated with membrane. Compressed helium of grade 4.5
plasmids), RNAi (dsRNA coating), and (99.995%) or higher should be used; lower
staining (lipophilic dye coating). Biolistic grades may contain contaminating material
delivery is fast, contact free, and can, in which can obstruct gas flow within the Helios
principle, be performed with minimal damage. Gene Gun as well as contaminate the
In addition, deep tissue layers can be reached biological sample. A helium tank pressurized
independently of tissue properties other than its to 2,600 psi [approximately 5 ft (1.7 m) high,
mechanical rigidity. Gold carrier particles are 291 cu ft standard in the United States] is
commercially available in a variety of sizes recommended, although a smaller tank [~2.5 ft
(0.5 1.5 um) and with surface coatings (~0.8 m) high] may be used. Follow all safety
instructions provided by the helium supplier
optimized for DNA, RNA, or lipophilic dyes.
for helium tank installation.
Requirements for System Operation
Operation of the Gene Gun System
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Loading Ratio (DLR). Typical DLRs range


between 1 and 5 g DNA/mg gold. Adding
Before the Bombardment
more DNA tends to cause agglomeration of the
1. Coat microcarriers with DNA, load into gold particles, probably as a result of DNA
tubes, and prepare cartridges prior to binding to more than one particle. The amount
of microcarriers delivered per target is referred
day of
to as the Microcarrier Loading Quantity
Experiment.
2. Check helium supply (50 psi in excess (MLQ). Typical MLQs range from 0.25 to 0.5
mg/cartridge for in vivo delivery to epidermal
of desired delivery pressure).
3. Clean and/or sterilize the Gene Gun, cells, but may be slightly lower for in vitro
tube holders, and barrel liners as delivery to mammalian cells.
appropriate.
4. Connect the Gene Gun to a helium MLQs and DLRs.
source.
5. Activate the Gene Gun: turn on the Procedure 1: Determining the Microcarrier
flow of helium to the desired pressure Loading Quantity (MLQ)
and with an empty cartridge holder in
place, make 23 pre-shots by 1. For most systems, delivering 0.5 mg of
gold per target is a good starting point.
engaging the safety interlock and firing
2. A 1 ml suspension will fill an 8.5" length
the trigger.
of tubing; one cartridge is 0.5" long. Each
30" length of tubing can be filled with
After the Bombardment
approximately 25" (3.0 ml) of DNA/gold
suspension. (There will be a void space at
1. Remove cartridge holder from Gene
each end.)
Gun.
2. Remove cartridges from cartridge 3. For delivering 0.5 mg of microcarriers per
target
(MLQ=0.5),
resuspend
the
holder.
DNA/microcarrier sample at 8.5 mg of
3. Turn off the helium pressure to the
gold/ml ethanol. A 25" length of tubing
system.
will require 25 mg of gold resuspended in a
4. Turn
the
regulator
value
volume of 3 ml of ethanol.
counterclockwise to de-pressurize the
system.
5. Disconnect the helium hose and Gene Procedure 2: Determining the DNA Loading
Gun.
Ratio (DLR)
Calculating the Amounts of Gold and
Plasmid Required
Prior to precipitating DNA onto the
gold particles and loading them into the GoldCoat tubing, it is necessary to calculate the
amount of DNA and gold required for each
transformation. Points to consider in making
these calculations are presented below. The
amount of DNA loaded per mg of
microcarriers is referred to as the DNA

1. For many applications, delivery of 1 g of


plasmid per target is a good starting point.
2. At a MLQ of 0.5 mg/cartridge, a DLR of 2
g DNA/mg gold results in loading 1 g of
DNA/cartridge and in delivery of 1 g of
DNA per target. Preparation of two lengths
of Gold-Coat tubing requires 100 g of
DNA and 50 mg of gold. The concentration
of DNA should be approximately 1 g/l
and the volume of DNA should not exceed
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the volume of spermidine in Section 5.2, 2. In a 1.5 ml microfuge tube, weigh out gold
microcarriers. (Refer to Procedure 1 for a
Precipitation of DNA onto Microcarriers,
detailed description on determining MLQ.
3. If the DNA is too dilute, concentrate it by
Refer to Table 2 for suggestions on the
ethanol precipitation. If a high DLR is
relative amounts of gold and microcarriers
desired, increase the volume of spermidine
required and on the length of tubing
and CaCl2 so that equal volumes of each
produced.)
component are added (spermidine, DNA,
and CaCl2) up to a total volume of 1,200 3. To the measured gold, add 100 l of 0.05
M spermidine. (However, if the volume of
l.
plasmid to be added in step 5 is greater
4. For a detailed description on determining
than 100 l, refer to the discussion above
which MLQs and DLRs will work best
for Procedure 2: Determining the DNA
for several mammalian targets.
Loading Rate, and add the appropriate
volume of spermidine.
Precipitation of DNA onto Microcarriers
4. Vortex the gold and spermidine mixture for
a few seconds then sonicate for 35
It is important to use an unopened
seconds using an ultrasonic cleaner to
bottle of 100% ethanol each day this step is
break up gold clumps.
performed. Opened bottles of ethanol absorb
water and the presence of water in the tubing 5. To the gold and spermidine mixture, add
the required volume of plasmid to achieve
while drying will lead to streaking, clumping,
the desired DLR. (Refer to Procedure 2 for
and uneven coating of the microcarriers over
a detailed description on determining DLR.
the inner surface of the Gold-Coat tubing,
Refer to Table 2 for suggestions on the
resulting in poor or unusable cartridges. All
relative amounts of gold and microcarriers
ethanol solutions should be opened only briefly
required and on the length of tubing
when in use and kept tightly capped when not
produced.) For co-transfection of multiple
in use. Polyvinylpyrrolidone (PVP) serves as
plasmids, add each of the plasmids at this
an adhesive during the cartridge preparation
step. DNA does not associate with the
process. At higher discharge pressures,
microcarriers prior to addition of CaCl2.
preparing cartridges with PVP can increase the
6. Mix DNA, spermidine and gold by
total number of particles delivered. The
vortexing ~5 sec.
optimum amount of PVP to be used must be
7. While vortexing the mixture at
determined
empirically.
Typical
PVP
moderate rate on a variable speed
concentrations range from 0.01 to 0.1 mg/ml.
vortexer, add 100 l of 1 M CaCl2
dropwise to the mixture. The volume
Protocol
added should equal that of the
spermidine in Step 3.
1. Prepare a stock solution of 20 mg/ml PVP
8. Allow the mixture to precipitate at
in ethanol in a small screw-cap container.
room temperature for 10 min.
Dilute this solution with ethanol to prepare
9. Most of the gold will now be in the
PVP solutions at the desired concentration
pellet, but some may be on the sides of
(generally 0.010.1 mg/ml); prepare 3.5 ml
the tube. The supernatant should be
of the dilute solution for each 30" length of
relatively clear. Spin the microcarrier
Gold-Coat tubing, (25 to be coated) in the
solution in a microfuge ~15 sec to
Tubing Prep Station. Keep these solutions
pellet the gold. Remove the supernatant
tightly capped when not in use. Prepare
and discard.
solution daily.
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10. Resuspend the pellet in the remaining


supernatant by vortexing briefly. Wash
the pellet three times with 1 ml of fresh
100% ethanol each time; spin ~5 sec in
a microfuge between each wash.
Discard the supernatants.
11. After the final ethanol wash, resuspend
the pellet in 200 l of the ethanol
solution containing the appropriate
concentration of PVP prepared in step
1. Transfer this suspension to a 15 ml
disposable polypropylene centrifuge
tube with a screw cap. Rinse the
microfuge tube once with 200 l with
the same ethanol/PVP solution and add
to the centrifuge tube. Add the
necessary volume of the ethanol/PVP
solution to the centrifuge tube to bring
the DNA/microcarrier solution to the
desired MLQ.
12. The suspension is now ready for
preparation.
Alternatively
the
DNA/microcarrier suspensions can be
stored for up to 2 months at -20 C.
Prior to freezing, tighten the cap
securely and put Parafilm around the
cap of the tube. After storage at -20 C,
allow the particle suspension to come
to room temperature prior to breaking
the Parafilm seal.

vortexer to resuspend and disrupt


agglomerated particles
2. Remove 50uL (3mg) of microcarriers to a
1.5mL microfuge tube
3. While vortexing vigorously, add in order:
5uL DNA (1ug/uL)-50ug CaCl2 (2.5M)
20uL spermidine (0.1M).
4. Continue vortexing for 2-3min;
5. Allow the microcarrier to settle for 1min;
6. Pellet the microcarriers by spinning for 2s
in a microfuge;
7. Remove the liquid and discard;
8. Add 140uL of 70% ethanol without
disturbing the pellet;
9. Remove the liquid and discard;
10. Add 140uL of 100% ethanol without
disturbing the pellet;
11. Remove the liquid and discard
12. Add 48uL of 100% ethanol
13. Gently resuspend the pellet by tapping the
side of the tube several times, and then by
vortexing at low speed for 2-3s
14. Remove six 6uL aliquots of microcarriers
and transfer them to the center of a
macrocarrier. An effort is made to remove
equal amounts (500ug) of microcarriers
each time and to spread them evenly over
the central 1cm of the macrocarrier using
the pipette tip. Desiccate immediately.
DNA delivery to target tissue

Loading the DNA/Microcarrier Suspension


into Gold-Coat

1. Once the microcarrier complex is


prepared then it is placed on
macrocarrier plateof gene gun and
ready for use.
2. Touch the target area with the spacer so
that the spacer is flush and the Gene
Gun is perpendicular to the target
surface. Activate the safety interlock
switch and press the trigger button to
deliver the DNA/microcarriers to the
target.
3. Repeat the shoot until all the areas are
shot.

The following procedure is sufficient for 6


bombardments; if fewer bombardments are
needed, prepare enough microcarriers for 3
bombardments by reducing all volumes by
1/2. When removing aliquots of microcarriers,
it is important to vortex the tube containing the
microcarriers
continuously in
order
to
maximize uniform sampling.
1. Vortex the microcarriers prepared in 50%
glycerol (60mg/mL) for 5min on a platform

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Summary of protocol for the gene gun


transfection:
Prepare gold or tungsten particles

Prepare DNA-coated particles

Place a macrocarrier

ISSN 2320-6020

Suicide
Gene
Therapy: This
application of the Gene Gun has been
used in the treatment of Cancer
patients. A gene that expresses a toxic
protein but has tumor specific
promoters is introduced to tumor cells.
When the protein is expressed the
tumor cell dies. The protein is only
toxic to tumor cells because the specific
promoters needed for expression are
only produced in tumor cells.
Immunomodulation: This method is
also used to fight Cancer. Using the
Gene Gun, a protein that will only be
expressed in tumor cells but will also
elicit an increased immune response are
inserted. An increased immune
response directed towards tumor cells
is obviously a desired effect.

Load on Gene Gun

Shoot

Screening
Fig:1.2 Summary of Protocol
Applications
Genetic
Vaccination: Genes
are
introduced into the body using the
Gene Gun with the purpose of eliciting
an immune response to the proteins
expressed by the delivered gene. This
method of vaccination may be safer
than other methods because only
foreign DNA in introduced and not
foreign proteins or killed vaccines.

Genetic Pharmacology: The Gene


Gun can be used to introduce genes that
will produce proteins that are useful or
therapeutic to an organism. Some
examples of this may be clotting factors
in hematologic disorders or increased
production of red blood cells in
organisms that are anemic. Sustained
expression of the introduced genes is a
problem in many cases often requiring
multiple administrations.
A Research Tool: The gene gun can be
used to insert promoters that will lead
to the expression of certain genes. The
effects of the amplification of certain
proteins is a valuable way for
researchers to study the functions of
these proteins
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Conclusion
Biolistic mediated gene transfer is very
efficient method of gene transfer its applicable
all type of host but it get more success in Plant
kingdom. In this method we dont need to
protoplast the plant cell we used it directly
with cell wall. With the help this method
researchers can able to engineer transformed
cell organelles such as chloroplast and
mitochondria. The method of gene gun can
also be used to make genetic modification in
the plant for e.g. plant can made resistant to
drought by making chages in their genetic
makeup or inserting new genes. Similarly
nutrional value can also be increased with help
thos technology.This method also show
promising sign in DNA vaccination, Gene
therapy, Transgenesis (Geneticaly modified
organism)
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