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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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Place a macrocarrier
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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.
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.
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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)
References
1. Albertini, M. R., Emler, C. A., Schell,
K., Tans, K. J., King, D. M. and
Sheeby, M. J., Cancer Gene Ther., 3, In
press (1996).
2. Andree, C., Swain, W. F., Page, C. P.,
Macklin, M. D., Slama, J., Hatis, D.
and Eriksson, E., Proc. Natl. Acad. Sci.
USA, 91, 12188-12192 (1994).
3. Armaleo, D., Ye, G. N, Shark, K. B.,
Sanford, J. C. and Johnston, S. A.,
Curr. Genet. 17, (1990).
4. Biewenga JE, Destree OH, Schrama
LH. Plasmid-mediated gene transfer in
neurons using the biolistics technique. J
Neurosci Methods 1997.
5. Boynton, J. E., Gillham, N. W., Harris,
E. H., Hosler, P. J., Johnson, A. M.,
Jones, A. R., Randolph- Anderson, B.
L., Robertson, D., Klein, T. M., Shark,
K. B. and Sanford, J. C., Science 240,
(1988).
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