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Biolistic ® PDS-1000/He Particle Delivery System Demonstration

Introduction Biolistic particle delivery is a method of transformation that uses helium pressure to introduce DNA-coated microcarriers into cells. Microprojectile bombardment can transform such diverse targets as bacterial, fungal, insect, plant, and animal cells and intracellular organelles. Particle delivery is a convenient method for transforming intact cells in culture since minimal pre- or post-bombardment manipulation is necessary. In addition, this technique is much easier and faster to perform than the tedious task of microinjection. Both stable and transient transformations are possible with the Biolistic particle delivery system. The Biolistic PDS-1000/He instrument uses pressurized helium to accelerate sub-cellular sized microprojectiles (gold or tungsten) coated with DNA (or other biological material) over a range of velocities necessary to optimally transform many different cell types. The system consists of the bombardment chamber (main unit), connective tubing for attachment to vacuum source, and all components necessary for attachment and delivery of high pressure helium to the main unit (helium regulator, solenoid valve, and connective tubing).

Figure 1. Biolistic components.

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Figure 2. Bombardment chamber components Power Switch, ON/OFF Controls supply of line electrical power to the instrument.

Vac/Vent/Hold Switch Controls application of vacuum to bombardment chamber. Vac applies vacuum from line source. Vent releases vacuum using filtered air. Hold maintains vacuum by isolating chamber. Bombardments should be performed with this switch in “Hold” position.

Fire Switch Controls flow of helium into Gas Acceleration Tube by activating Solenoid Valve. Illuminated red when enabled. Fire Switch must be held ON continuously until Rupture Disk bursts; then release Fire Switch to stop flow of helium.

Vacuum Gauge Indicates level of vacuum in bombardment chamber.

Vacuum/Vent Rate Control Valves

Regulate rate of application and relief of vacuum in bombardment chamber. Clockwise rotation closes valves.

Helium Pressure Gauge Indicates helium pressure in Gas Acceleration Tube.

Bombardment Chamber Door Closes chamber with a solid piece of polycarbonate plastic.

Rupture Disk Retaining Cap Seals Rupture Disk against chamber end of Gas Acceleration Tube. This must be tightened securely. The Torque Wrench is used in the holes in the cap.

Microcarrier Launch Assembly Holds the DNA/microcarrier preparation on a Macrocarrier sheet over the Stopping Screen in the path of the helium shock wave.

Target Shelf Holds the biological target in a Petri plate in the path of the accelerated DNA/microcarrier preparation. Particle flight distance is determined by positioning the shelf at one of four levels using slots in the chamber walls.

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The Biolistic Process The Biolistic PDS-1000/He system uses high-pressure helium, released by a rupture disk, and partial vacuum to propel a macrocarrier sheet loaded with millions of microscopic tungsten or gold microcarriers toward target cells at high velocity. The microcarriers are coated with DNA or other biological material for transformation. The macrocarrier is halted after a short distance by a stopping screen. The DNA-coated microcarriers continue traveling toward the target to penetrate and transform the cells. The launch velocity of microcarriers for each bombardment is dependent upon the helium pressure (rupture disk selection), the amount of vacuum in the bombardment chamber, the distance from the rupture disk to the macrocarrier macrocarrier gap distance (A), the macrocarrier travel distance to the stopping screen (B), and the distance between the stopping screen and target cells target distance (C).

Figure 3. The Biolistic bombardment process.

Figure 4. Components of the rupture disk retaining cap (left) and microcarrier launch

assembly (right).

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In Figure 3, A, B and C are all adjustable distances that influence the velocity with which the microcarriers hit the target cells. The most important factors that must be optimized in Biolistic transformation are target distance and microcarrier size. All parameters must be optimized empirically. Table 1 shows the effect of some of these parameters in transformation.

Table 1. Particle accelerator parameters that affect transformation with the PDS-1000/He.

Parameter Observed Effect on transformation

Microparticle size Major Target distance Major Vacuum Major Helium pressure Variable Gap distance Minor Macrocarrier travel distance Minor

When optimizing your system start with the conditions given in table 2, then examine in order: target distance, microparticle size and type and helium pressure.

Table 2. Suggested starting conditions for optimizing several types of biological systems using the PDS-1000/He system.

Biological system Microcarrier Vacuum

Helium pressure Target distance

Bacteria 0.8 µm tungsten

29 1,100 6 cm

Yeast 1.1 µm tungsten

28 1,100 6 cm

Plant 1.2 µm tungsten

28 1,100 9 cm

Organelles 1.0 µm tungsten

28 900 6 cm

In all cases the rupture disk-macrocarrier gap distance was set at ¼ inch and the macrocarrier travel distance at 8 mm. To optimize Biolistic transformation, begin with the parameters given in Table 2 and test both above and bellow those listed.

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Procedure for the transformation of Cauliflower During this demonstration we will bombard cauliflower tissue with the plasmid pCAMBIA 2202-sGFP S65T that contains the green fluorescent protein (GFP) gene to observe transient expression of this gene. Equipment Materials Biolistic PDS-100/He Scalpel Vortex Microcentrifuge 1.5 ml microfuge tubes∗ 25 and 50 ml beakers* Pipette and tips* Petri dishes Macrocarriers* Macrocarrier holders* Rupture disk retaining cap* 900 dpi rupture disks Stopping screens*

Cauliflower Microparticles (Tungsten M-17, 1.1 micron) 100% ethanol (HPLC or spectophotometric grade) 70% ethanol Distilled water* 50% glycerol* Purified plasmid DNA (1 µg/µl) 2.5 M CaCl2!

0.1 M spermidine (free base, tissue culture grade)! Filter paper Desiccant CaCl2 70% isopropanol

∗ Should be sterilized by autoclaving ! Should be filter sterilized

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A) Microcarriers The following procedure prepares tungsten or gold microcarriers for 120 bombardments using 500 µg of the microcarrier per bombardment, based on the method of Sanford, et al. [Methods in Enzymology, 217, 482-509 (1993)].

1. Weigh out 30 mg of microparticles into a 1.5 ml microfuge tube. 2. Add 1 ml of 70% ethanol (v/v). 3. Vortex vigorously for 3–5 minutes (a platform vortexer is useful). 4. Incubate for 15 minutes. 5. Pellet the microparticles by spinning for 5 seconds in a microfuge. 6. Remove and discard the supernatant. 7. Repeat the following wash steps three times:

a. Add 1 ml of sterile water. b. Vortex vigorously for 1 minute. c. Allow the particles to settle for 1 minute. d. Pellet the microparticles by spinning for 2 seconds in a microfuge. e. Remove the liquid and discard.

8. After the third wash, add 500 µl sterile 50% glycerol to bring the microparticle concentration to 60 mg/ml (assume no loss during preparation).

The microparticles can be stored at room temperature for up to two weeks. Tungsten aliquots should be stored at -20 °C to prevent oxidation. Gold aliquots can be stored at 4 °C or room temperature. Store dry tungsten and gold microcarriers in a dry, non-oxidizing environment to minimize agglomeration. B) Coating Washed Microcarriers with DNA The following procedure is sufficient for six bombardments. If fewer bombardments are needed, adjust the quantities accordingly. Note that the macrocarrier/macrocarrier holders should be assembled (see section C) and autoclaved before starting this step. The rupture disk retaining cap should also be autoclaved.

1. Vortex the microcarriers prepared in 50% glycerol (60 mg/ml) for 5 minutes on a platform vortexer to resuspend and disrupt agglomerated particles.

2. Remove 50 µl (3 mg) of microcarriers to a 1.5 ml microcentrifuge tube. When removing aliquots of microcarriers, it is important to continuously vortex the tube containing the microcarriers to maximize uniform sampling. When pipetting aliquots, hold the microcentrifuge tube firmly at the top while continually vortexing the base of the tube.

3. While vortexing vigorously, add in order: a. 5 µl DNA (1 µg/µl) b. 50 µl 2.5 M CaCl2 c. 20 µl 0.1 M spermidine (free base, tissue culture grade)

4. Continue vortexing for 2–3 minutes. 5. Allow the microcarriers to settle for 1 minute. 6. Pellet microcarriers by spinning for 2 seconds in a microfuge.

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7. Remove the liquid and discard. 8. Add 140 µl of 70% ethanol (HPLC or spectrophotometric grade). 9. Remove the liquid and discard. 10. Add 140 µl of 100% ethanol. 11. Remove the liquid and discard. 12. Add 48 µl of 100% ethanol. 13. Gently resuspend the pellet by pipetting up and down a few times, and then by

vortexing at low speed for 2–3 seconds. C) Loading DNA-coated microcarriers onto a macrocarrier/macrocarrier holder

1. Assemble the macrocarriers onto the macrocarrier holders and sterilize by

autoclaving.

2. Place the macrocarrier/macrocarrier holder into a desiccating chamber. A

small desiccating chamber consists of a sterile 60 mm tissue culture Petri dish (with lid) containing CaCl2 as desiccant in the base of the dish (Figure 5). The desiccant is covered with a small piece of filter paper to provide a stable platform for the macrocarrier/macrocarrier holder. The sterile macrocarrier/macrocarrier holder is placed atop the filter paper, with the macrocarrier facing up and the stainless steel holder touching the filter paper.

3. For each macrocarrier, remove 6 µl aliquots (approximately 500 µg) of microcarriers and spread evenly over the central 1 cm of the macrocarrier using a pipette tip. Pipette from a continuously vortexed tube and rapidly apply suspended microcarriers to the macrocarrier, as microcarriers quickly settle out from the ethanol solution in the tube or even in the pipette tip.

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4. Immediately cover the culture dish after application of the microcarrier suspension to the macrocarrier. The ethanol should evaporate within 10 minutes to leave the DNA-coated micro-carriers adhering to the macrocarrier. The loaded macrocarriers should be used within 2 hours.

D) Plant tissue

1. Cut the main, white stem of a cauliflower into sections of approximately 1 cm2 and 2-4 mm thick.

2. Place 4 of the segments in the center of a petri dish on top of a filter paper moistened with distilled water and keep covered until needed.

E) Performing the bombardment

1. Verify that the pressure of the helium tank is set at 1100 dpi (200 dpi above the rupture pressure of the rupture disks).

2. Assemble the rupture disk retaining cap with a 900 dpi rupture disk. Before placing the rupture disk, briefly wet it in 70% isopropanol.

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3. Set up the macrocarrier launch assembly putting in the center of the brass adjustable nest in the following order (from bottom to top):

a. Stopping screen b. Microcarrier/microcarrier holder c. Microcarrier cover lid

4. Place the macrocarrier launch assembly on the top shelf of the

bombardment chamber. 5. Place the petri dish with the cauliflower segments (without the lid) on

the target plate on the third shelf (6 cm bellow the macrocarrier assembly).

6. Close the bombardment chamber door. 7. With the power ON, start the vacuum pump and turn the central switch

to VAC. 8. When the vacuum gauge shows 23 in Hg turn the switch to HOLD. 9. Press the Fire switch continuously until the rupture disk bursts and the

helium pressure gauge drops to zero. 10. Turn the central switch to VENT to release the vacuum. 11. Open the bombardment chamber door and remove your tissue from

the chamber. Cover the petri dish.

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12. Unload the macrocarrier and stopping screen from the macrocarrier launch assembly.

13. Unload the spent rupture disk. 14. Keep the cauliflower segments in the dark for 24-48 hrs before

observing under the microscope with blue light.