Note: Descriptions are shown in the official language in which they were submitted.
WO 94/17195 PC'TlUS93100817
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Title: PRETREATMENT OF MICROPROJECTILES PRIOR TO
USING IN A PARTICLE GUN
BACKGROUND OF THE INVENTION
This invention relates to an improved process for
transferring biological materials such as nucleic acid
into the cytoplasm of living cells. With the rapid
advancement of recombinant DNA technology, there is a
wide-ranging need for biologists to transfer biologic
substances from one cell to another, and to transfer
synthetic biological material into living cells to
exert their activity therein. Such materials can
include biological stains, proteins (antibodies or
enzymes), and, most commonly, nucleic acids genetic
material (either RNA or DNA). Most of the techniques
used are painstakingly slow and use methods which
transport materials into, at most, only a few cells at
a time. More recently, there has been developed a
particle bombardment process which utilizes a particle
gun, as described in Sanford, et al., 1987, "Delivery
of Substances Into Cells and Tissues Using A Particle
Bombardment Process," Journal of Particle Science and
TechnoloQV 5:27-37,
An earlier invention of one of the joint
inventors, Dwight Tomes, relates to an improved
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particle gun which uses a gun having a rifled barrel.
The disclosure of particle gun bombardment and the
method of transport of biological materials such as
DNA into living cells is described in Tomes, IMPROVED
PARTICLE GUN, filed May 12, 1989, Serial No.
07/351,075,
The effectiveness of particle transport is, of
course, measured by the ability of living cells into
which the transported particles have been inserted to
pick up and express.the biological material. This, of
course, depends upon a wide variety of conditions.
The less the expression, the less successful the
transport. Correspondingly, the more successful the
expression of the living cells, i.e. the extent that
they pick up and express the transported biological
material, the better the nucleic acid insertion
experiments.
In the particle gun technique the biological
material (DNA for example) is mixed with the carrier.
The carrier generally is comprised of a substantially
inert metal in the form of small beads which function
as microprojectiles. Generally, the microprojectiles
have a diameter within the range of about 1 micron to
about 4 microns. These beads can be made from
tungsten, palladium, platinum or gold or an alloy
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thereof. Tungsten is preferred for reasons of
economics. However, tungsten generally does not give
as good or as successful transport and expression as
does gold unless used with the parent pre-treatment
process. Beads can generally range from about 1
micron to about 1.5 microns in diameter.
In the most preferred process, the beads are mixed
with a small amount of biological material such as DNA
or RNA. This is mixed with calcium chloride and a
certain amount of polyamine is added.
Generally the ranges of each of these ingredients
should be as follows:
Twenty-five ul of tungsten particles at a
concentration of 15-400mg in 2m1 sterile~water are
placed in a sterile 10 ml centrifuge tube and agitated
to suspend the beads. The preferred amount of beads
is 375mg/2m1. Twenty-five ul of the suspended
tungsten beads are placed in an Eppendorf tube and DNA
is added at a concentration of 1 ug/ul with the amount
varying between lul and 20u1, the preferred amount
being 10u1. A calcium chloride solution of 25/pl and
having a concentration between 1.0-4. OM, preferably
2.5M is mixed with the DNA/bead mixture.
While addition of spermidine to the biological
material/microprojectile combination has been
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previously employed, it has now been more broadly
discovered that addition of a variety of polyammines
to mixtures of tungsten beads and DNA or RNA in
preparing microprojectiles significantly improve
rates of transformation. While not intending to be
limited by theory, it is believed that the polyamine
improves delivery of biological material to the cells
in this process by improving adherence of the
materials to the microprojectile beads. Suitable
polyammines have been found to include, for example,
spermine, spermidine, caldine, thermine, and the like.
The preferred polyamine, spermine, has been found to
be superior to the previously disclosed spermidine
additive. Accordingly, at this point a polyamine,
preferably spermine, in an amount of 10 pl and a
concentration between 0.05M and 0.5M, preferably at
O.1M is added followed by finger vortexing. This
mixture is allowed to stand for 10 minutes prior to
centrifugation for one to two minutes at 9,000 rpm.
Centrifugation may be used but is not required. The
microprojectile mixture forms a pellet at the bottom
of the Eppendorf tube. Before use, supernatant is
withdrawn from the tube to provide a final volume of
30u1.
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The DNA/bead mixture is sonicated briefly to
suspend the microprojectiles prior to use. The
suspended microprojectiles carrying the biological
material are transferred to the forward end of the
macroprojectile by micropipette in aliquots of 1-5p1,
with 1.5u1 preferred-:
Among the difficulties which have been encountered
in the past in using microprojectiles is that large
masses of DNA would clump onto the particles which
make it harder to separate and also make it more
likely that the particles will kill the cells during
bombardment.
As can be seen, there is therefore a continuing
need to develop microprojectile bombardment processes
for improved transformation of biological materials
into living cells by a process which will not kill the
cells during bombardment, by a process which can
utilize inexpensive tungsten beads, and by a process
can use such lesser expensive beads and still achieve
a highly successful transformation and expression.
This invention has as its primary objective the
fulfillment of this need.
SUMMARY OF THE INVENTION
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The invention relates to an improvement in the
process of successfully transporting biological
material into living cells by bombarding the cells
with biological material coated particles. The
preferred particles are tungsten and the method of
the present invention allows use of the more
economical tungsten beads rather than gold. The
achievement of the objectives of the present
invention is accomplished by pretreating the
preferred tungsten beads with a strong inorganic acid,.
preferably nitric acid.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the process of this invention
prior to coating of the microprojectile beads, they
are first pretreated with a strong inorganic acid.
The strong inorganic acid is selected from the group
consisting of nitric acid, sulfuric. acid, hydrochloric
acid and phosphoric acid or mixtures thereof. The
preferred acid is nitric acid.
The concentration of the strong inorganic acid is
not critical. At the lower end, a concentration of as
low as .O1 molar has been used successfully and at the
upper end there appears to be only a process economic
consideration. Namely excessive amounts are not
f~
WO 94/17195 < < PCT/US93100817
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harmful to the metallurgy or surface of the beads, but
there is not reason to use concentrations beyond an
amount sufficiently concentrated to effectively clean
and pretreat the beads. Generally, the upper limit
would be a practical limit of about 1.0 molar,
preferably the concentration of the acid would be
within the range of 0.1 molar to about 0.5 molar.
The time of treatment is not critical but,
generally can range from about 5 minutes to about 60
minutes, preferably from about 10 minutes to about 20 .
minutes. The importance of time is sufficient time
for the beads, to successfully contact the nitric
acid. In this regard, successful contact is best
achieved when beads are agitated during their pre-
treatment. Preferably the agitation is continuous
and most preferably the agitation is by sonication.
Following the sonication procedure, the particles, if
treated with nitric acid, should not settle out of the
liquid suspension if they are clean. Following a 20
minute sonication, the washed particles stayed in
suspension, thus it was determined that the 20 minute
wash was sufficient for successfully cleaning the
tungsten particles. Prior to the nitric acid wash,
tungsten immediately fell out of suspension.
WO 94!17195
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The precise beads employed are not critical and
the process shows distinct advantages when the beads
are any beads selected from the group consisting of
tungsten, palladium, platinum~and gold or an alloy
thereof. Preferably the beads have a diameter of from
about 1 micron to about 1.5 micron. The most
preferred beads are tungsten since by using the pre-
treatment of the present invention tungsten can be
used as effectively as more expensive metals such as
platinum, palladium and gold.
After the pretreatment process of the present
invention a variety of conventional steps may be
employed. The acid is rinsed from the beads, and the
rinse may be a water rinse, for example followed by an
alcoholic rinse, for example, followed by ethanol.
The beads are then conventionally air dried, perhaps
by vacuum speed drying. After this, the biological
material such as DNA is added to the microprojectiles
and proper mixing is done. Conventional additives is
know to be mixed with a biological material such as
calcium chloride and a certain amount of amines such
as a polyamine. Thereafter the plant tissue is
bombarded to achieve the transport of biological
material into living cells or simulation therein.
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The following examples are offered to illustrate
but not necessarily limit the present invention. They
demonstrate that with the pretreatment of the present
invention tungsten is more effective than gold beads.
EXAMPLE 1
In this example a particle gun experiment tested
tungsten and gold after nitric acid was used to clean
the surfaces of the particles as a means to reduce
flocullation and increase DNA binding.
The purpose was to evaluate the effect of a nitric
acid cleaning on tungsten and gold particles prior to
drying them in a speed vacuum. They were compared to
a standard control.
The genotype material used was an embryonic maize
suspension of DNA. The maize suspension medium
contained Murashige and Skoog salts: Reference:
Murashige, T., Skoog, F.: A Revised Method for Rapid
Growth and Bioassays with Tobacco Tissue Cultures,
Physiol. Plant. 15:473-497 (1962) with 2.Omg/L of
2,4-D, which is 2,4-Dichorophenoxy-acetic Acid and 3$
sucrose. In addition, the media used involved maize
callus medium with the following description: AT
salts with 700mg/L proline, 0.75mg/L 2,4-D, 3$
sucrose. Reference: Tomes, D., Cell Culture,
WO 94117195 PC'TIUS931008I7
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Somatic Embryogenesis and Plant Regeneration in Maize,
Rice, Sorghum, and Millets in Cereal Tissue and Cell
Culture, Nijnoff and Junk, Amsterdam, 1985.
The particle gun treatment involved use of the particle
gun as described previously in the Tomes application.. The
beads were tungsten l.8um from GTE, tungsten l.2um
from GE, gold (flakeless) from Engelhard Industries,
25mg tissue per plate being used. There was a 0.25M
mannitol pretreatment, overnight, and there was one
bombardment per sample using pDP460 DNA which
contains the GUS gene. pDP460 is described in Example
2 below.
The bombardment occurred in the following way.
Suspension cells were used one day after subculture
and sieved through a 710um screen and resuspended in
maize suspension medium + 0.25M mannitol at 50mg/ml
(lg tissue in 20m1 medium). They were agitated on the
shaker overnight. To ready for particle gun
treatment, 0.5m1 aliquots were pipetted onto the
center of double layers of 617 Whatman grade filter
paper to which lml maize suspension medium + 0.25M
mannitol has been added. The cells were concentrated
in the center of each plate to maximize exposure to
bombardment.
n
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Six repetitions were used for all particle
treatments including six repetitions for the standard
positive control. Twenty-six samples in total were
used. Two independent repetitions of the experiment
were completed.
To clean the tungsten and gold particles, we
weighed 375mg particles in 2m1 O.1M nitric acid. Then
we sonicated on ice for 20 minutes using the sonicator
set at the minimum to keep the particles suspended.
Next we rinsed particles two times using sterile
deionized water; on final rinse, we replaced with 1 ml
of 95~ etOH. This was followed by speed vacuum
drying.
All samples received one bombardment by the
particle gun.
Following particle gun treatment, we transfered
the top filter paper of each sample to maize callus
medium. The cells were incubated at 28° Celsius in
the dark for 48 hours and then assayed for transient
GUS activity.
Forty-eight hours post treatment all samples were
sacrificed for the GUS cytochemical assay. Based on
transient GUS activity, we compared particle types,
particle cleaning and particle sources.
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An analysis of variance showed significant
differences between microprojectile type (gold,
tungsten) and between microprojectile washing
treatments in this experiment, however, no
statistically significant interaction overall was
found between these variables. Using a t-test to
analyze each microprojectile type and treatment
separately, the only significance found was with the
GTE tungsten. This is what was expected as there was
such a big discrepancy in performance between tungsten
from GTE versus GE. After the nitric acid treatment,
the performance of both sources of tungsten are nearly
equal.
The following table shows the data for the present
tests.
TABLE 1
Beta-glucuronidase cytochemicalanalysis for transient
gene expression com pleted 36
hours post
treatment.
Genotype: 54-68-5, maize embryogenic
suspension
DNA: pPHI460
One bombardment per sample
Particle Particle GUS Cell Groups
Type Treatment DNA N Min Mean Max
Tungsten, GE EtOH 460 12 29 137 342
Tungsten, GE Nitric Acid 460 12 62 217 460
Significance = 0.111
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(Table 1 Cont.)
Particle Particle GUS Cell Groups
Type Treatment DNA N Min Mean Max
Tungsten, GTE EtOH 460 11 3 81 169
Tungsten, GTE Nitric Acid 460 12 39 187 440
Significance 0.023
=
Gold Flakeless EtOH 460 12 4 53 122
Gold Flakeless Nitric Acid 460 12 8 100 259
Significance = 0.119
* Significance of particle treatment within particle
type was measured using a t-statistic for a small
sample size.
The data in Table 1 as described illustrates that
first cleaning surfaces of the tungsten particles is
an effective means to reduce flocculation and increase
DNA binding, in comparison of use in non-nitric acid
treated particles in comparison with gold treated or
nontreated particles.
EXAMPLE 2
In this example, the process of the present
invention, using tungsten pretreated with nitric acid
was compared with tungsten beads not pretreated with
nitric acid. It also compared with bombardment of
cells using non-nitric acid treated tungsten
microprojectiles in a particle gun, using the stan-
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dard protocol recommended and publicly published by
DuPont. The DuPont protocol, as published, is as
follows:
Weigh 60 mg particles and suspend in 1 ml 100
ethanol. Sonicate. Centrifuge at 10,000 RPM for one
minute to pellet particles. Withdraw supernatant of
ethanol and replace with 1 ml sterile deionized water.
Sonicate. Centrifuge at 10,000 RPM for one minute.
After final rinse, resuspend in 1 ml deionized water.
Store in the freezer. After preparation DNA coating
of the particles can be completed.
Prepare DNA-coated particles in small aliquots
within microtubes (each aliquot being enough for three
bombardments). Many aliquots can be made at one
time. For one aliquot, place 25p1 of the washed
particles (ensure complete resuspension) into a
microtube, then, sequentially: add 2.5u1 DNA (at
ug/ul) and finger vortex five times; add 25u1 of 2.5
CaCl2 and finger vortex five times; and 10u1 of
spermidine (0.1 M-free base) and finger vortex five
times. Let sit for about ten minutes. Spin down for
3 minutes, and discard 40 to 50u1 of the supernatant
(this will concentrate the particles and leave just
enough for 3 bombardments). Repeat this process for
all remaining aliquots.
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In this example, the DuPont protocol was
compared with the protocol of the present invention,
wherein tungsten particles, 1.8u particles, were
prepared in accordance with the following procedure:
Weight 375mg of 1.8u tungsten and suspend in 2m1
of O.1M nitric acid. Sonicate for 20 minutes on ice.
Centrifuge at 10,000 RPM for one minute and remove
nitric acid. Add 2m1 of sterile deionized water,
sonicate briefly then centrifuge, repeat water, rinse
two times. Remove water supernatent and add 2m1 of
100 Ethanol. Sonicate to resuspend particles, taking
out.25u1 aliquot following each sonication. Place
aliquot into individual 1.5m1 Eppendorf tubes. Speed
vacuum dry tungsten/ethanol suspension. Store
particles at room temperature, covered.
The Pioneer tungsten/DNA precipitation method is
as follows: add lOpl (at l~ag/pl) DNA to speed vacuum
dried tungsten, mix with pipettor. Add 25u1 of 2.5M
CaCl2 to tungsten/DNA suspension, mix with pipettor.
Add 10u1 to O.1M spermine to tungsten/DNA/CaCl2
suspension, finger vortex. Allow suspension to stand
at room temperature for 15 minutes, then withdraw 15u1
of supernatent. Sonicate suspension prior to
aliquoting 1.5u1 aliquots onto macroprojectiles.
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The embroyogenic maize suspension 2-122-4 (W23 x
B73) was used. The culture media and particle
bombardment procedures were as outlined in example 1
(see page 9).
The particle preparations prepared on day 1 by
the respective methods were sequentially sampled for
experiments on days 1, 2, 3, 4, and 5 using particles
on day 1. DNA/particle mixing using the respective
procedures was done on each day. Sixteen samples for
each set of this example were completed. Fourteen
were treated with pDP460 DNA. The plasmid pDP460
contains an enhanced promoter spanning nucleotides -
421 to +2 of CaMV 35S [R. C. Gardner et al. Nucleic
Acids Res. 9, 2871 (1981)], a 79-by Hind III-Sal I
fragment from pJII101 spanning the 5' leader sequence
of tobacco mosaic virus [D. R. Gallie, D.E. Sleat, J.W.
Watts, P.C. Turner, T.M.A. Wilson, Nucleic Acids Res.
15, 3257 (1987)], a 579-by fragment spanning the first
intron from maize Adhl-S [E. S. Dennis et al., ibid.
12, 3983 (1984)], and 1870-by fragment from pRAJ275
spanning the GUS coding sequence [R. Jefferson, S.
Burgess, D. Hirsh, Proc. Natl. Acad. Sci. U.S.A., 83,
8447 (1986)] and a 281-by fragment containing a
polyadenylation site from the Agrobacterium
tumefaciens nopaline synthase gene [M. Bevan, W.M.
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Barnes, M.D. Chilton, Science II, 369 (1983)] in pUCl8
[C. Yanisch - Perron, J. Vieira, J. Messing, Gene 33,
103 (1985)]. The remaining two samples were
nontreated controls. All treated samples received
one bombardment by the particle gun. Immediately
following bombardment all samples were transferred to
maize suspension medium and were maintained in the
dark at 28°C for 36 hours.
Thirty-six hours post treatment, all samples
were sacrificed for GUS cytochemical assay.
Individual cells which stain positive for GUS for both
the invention and for the DuPont protocol were
recorded. Overall, gene expression for each treatment
for each day was compared by establishing a ratio
between the invention process and the DuPont protocol,
with the DuPont protocol not showing any pretreatment
with nitric acid. The following Table 2 sets forth
the results.
WO 94117195 PCTIUS93100817
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Table 2
Treatment GUS individual cells Ratio
Day N Protocol Min Mean Max INV/DuPont
Day 1 7 Invention 339 452 572 1.7:1.0
7 DuPont 173 274 319
Day 2 7 Invention 207 258 292 1.8:1.0
7 DuPont 84 146 180
Day 3 7 Invention 148 211 301 2.9:1.0
7 DuPont 4 73 100
Day 4 7 Invention 228 380 485 3.5:1.0
7 DuPont 8 110 194
Day 5 7 Invention 62 217 389 2.4:1.0
7 DuPont 0 91 137
The data of significance in Table 2 is the data
under ratio of "INV" to DuPont. There, it is shown
that the GUS gene (pDP460) is successfully delivered
and expressed in the cells, as measured by the cells
turning blue in a much higher ratio, with the process
of the present invention than with the standard DuPont
process which utilizes similar tungsten particles,
but does not pretreat with nitric acid.
EXAMPLE 3
In this example, a comparison of the process of
the present invention using nitric acid pretreated
beads in the manner described in Example 2 and using
tungsten beads which were not pretreated according to
the standard DuPont protocol, were prepared precisely
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as above described. The example was run to show not
only successful transport into the cells, but whether
or not the DNA after transport is integrated into
plant DNA such that cell division carrying the foreign
DNA occurs. In other words, can you not only transfer
the DNA into the cells, but having done that, can you
successfully grow up a plant, which has the foreign
DNA integrated into the plant DNA. In this example,
standard procedure prenitric acid treated tungsten
beads were used with the plant material being Xanthi
tobacco cotyledons. The DNA expressed was pDP456
[NPTII+GUS]. The plasmid pDP456 contains two
different plant transcription units (PTU's). The
first PTU includes an enhanced promoter spanning
nucleotides -421 to +2 of CaMV 35S with the region
from -421 to -90 duplicated in tandem [R.C. Gardner et
al., Nucleic Acids Res. 9, 2871 (1981)], a 79-by Hind
III-Sal I fragment from pJII101 spanning the 5' leader
sequence of tobacco mosaic virus [D. R. Gallie, D.E.
Sleat, J.W. Watts, P.C. Turner, T.M.A. Wilson Nucleic
Acids Res. 15, 3257 (1987)], an 1870-by fragment from
pRAJ275 spanning the GUS coding sequence [R.
Jefferson, S. Burgess, D. Hirsh, Proc. Natl. Acad.
Sci. U.S.A. 83, 8447 (1986)] and a 281-by fragment
containing a polyadenylation site from the
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Agrobacteriiun tumefaciens nopaline synthase gene [M.
Bevan, W.M. Barnes, M.-D. Chilton, ibid. 11, 369
(1983)] in pUCl8 [C. Yanisch-Perron, J. Vieira, J.
Messing, Gene 33, 103 (1985)]. The second PTU is
identical to the first, except that it includes the
NPTII coding sequence [R. T. Frayley et al., Proc.
Natl. Acad. Sci. U.S.A. 80, 4803 (1983)] in place of
GUS. In accordance with this example four in vitro
grown Xanthi cotyledons were prepared and bombarded
as described in Tomes et al. Plant Mol. Biol. 14: 261-
268 (1990).
Two independent experiments were completed in
which cotyledons were bombarded for each micropro-
jectile/DNA protocol.
The microprojectile/DNA DuPont comparison
treatments were made using the earlier described
standard DuPont protocol. The treatments of the
present invention were as described in the previous
example. All DNA samples received one bombardment by
the particle gun. After particle gun treatment, all
DNA treated samples were transferred to a selection
medium as described in the Tomes, et al. publication.
The number of colonies recovered with each
treatment were recorded, and Table 3 indicates NPTII
enzyme assays confirmed stable integration of pH1456
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in each sample noted as NPTII+. Table 3 shows the
assay results for independent trans-genes from the
process protocol or the DuPont protocol.
Table 3
Sample DNA Protocol NPTII
25,C1 456 Invention +
52,C1 456 Invention +
52,C2 456 Invention -
20,C1 456 Invention +
26,C1 456 Invention +
26,C2 456 Invention -
26,C3 456 Invention +
26,C4 456 Invention +
55,C.1 456 Invention +
50,C1 456 Invention +
10,C1 456 DuPont +
12,C1 456 DuPont +
14,C1 456 DuPont +
14,C2 456 DuPont +
2,C1 456 DuPont +
41,C1 456 DuPont -
43,C1 456 DuPont +
34,C1 456 DuPont +
CONTROL
CONTROL
Out of all the samples treated with the process
protocol of the present invention and expendables, 800
of the cotyledons recovered stably transformed
colonies. From all samples treated with the DuPont
DNA protocol, 60 of the cotyledons recovered stably
transformed colonies. The process of the present
invention showed a higher number of transformation.
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EXAMPLE 4
Example 4 was run to confirm if nitric acid
treatment resulted in an increase in transformation
with the DuPont protocol compared with the protocol of
the present invention. In this example, the material
used was genotype 2-122-4 (W23 x 3) embryogenic maize
suspension. The DNA was pDP460. In the particle gun
treatments, the process of the present invention was
employed using a nitric acid treatment and compared
with the DuPont DNA/particle mixing protocol, and the
DuPont protocol using nitric acid as the only
variance. The methodology was as Example 1.
Forty samples were completed for this example.
They were treated with the pDP460 DNA and 20 samples
for each method were tested. All treated samples
received one bombardment by the particle gun.
The standard protocol for mixing DNA/particles
described in Example 2 and of the DuPont method as
described in Example 2 were then followed. Following
the particle gun treatment, all samples were
transferred to maize callus medium and maintained in
the dark at 28° C.
Thirty-six hours post-treatment, all samples
were sacrificed for GUS cytochemical assay. There-
after, the transient gene expression was compared
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between the two treatments based on the number of
visible blue stained cells present in each sample.
The data is summarized in the following Table 4.
Table 4
GUS individual cells Coefficient
Protocol N Min Mean Max of Variation
DuPont
Original 20 113 326.2 491 25.19%
Invention
Standard 20 203 430.9 728 29.61%
In Table 4 it can be seen that statistically
significant differences (letters denote statistical
difference at 95% confidence using analysis of
variance) were found between the DuPont protocol and
the protocol of the present invention in this
experiment. These results are in accord with earlier
experiments showing that indeed the nitric acid
pretreatment allows much higher transient gene
expression.
