Protocols for biolistic gene transformation (Bombardment)

Device Type: BIO-RAD PDS-1000/He Biolistic Particle Delivery System

Recommended starting particle size/type for bombardment of various cell types is

Bacteria 0.7 µm (M5) tungsten

Yeast 0.6 µm gold

Algae 0.6 µm gold

Plant cells/tissue 1.0 µm gold

Animal cell cultures 1.6 µm gold

Sub-cellular organelles 0.6 µm gold

Photos, figures and tables are from www.bio-rad.com website.

Procedures were developed by Sanford et al (1992)

1. Microcarrier preparation:

For 120 bombardments using 500ug per bombardment

1. In a 1.5mL epi, weigh out 60mg of microparticles;

2. Add 1mL of 70% ethanol, freshly prepared;

3. Vortex on a platform vortexer for 3-5min;

4. Incubate for 15min;

5. Pellet the microparticles by spinning 5s in a microfuge;

6. Remove the liquid and discard;

7. Repeat the following steps 3 times;

a. Add 1mL of sterile water;

b. Vortex for 1min;

c. Allow the particles to settle for 1min;

d. Pellet the microparticles by spinning for 2s in microfuge;

e. Remove the liquid and discard.

8. Add sterile 50% glycerol to bring the microparticle concentration to 60mg/mL (assume no loss during preparation);

9. Store the microparticles at r/t for up to 2wks.

2. Coating DNA onto microcarriers:

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 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).

(3 -> 25 -> 10)

(4 -> 32 -> 7)

(7.5 ->75 ->15)

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.

For BY2 cells, it is better to have the cells treated in 0.25M Sorbitol to make them a little “wilt”, then bombardment them;

For callus, it is relatively hard, so they can be bombarded 1-2 times, then inoculate with Agrobacteria;

Leaves are easy to be shot through;

Other tissues refer to related references or try-and-error way.

Performing a Bombardment

Quick Guide

Before the Bombardment

1. Select/adjust bombardment parameters for Gap distance between rupture disk retaining cap and microcarrier launch assembly. Placement of stopping screen support in proper position inside fixed nest of microcarrier launch assembly

2. Check helium supply (200 psi in excess of desired rupture pressure).

3. Clean/sterilize:

Equipment: rupture disk retaining cap, microcarrier launch assembly

Consumables: macrocarriers/macrocarrier holders

4. Wash microcarriers and resuspend in 50% glycerol

5. Coat microcarriers with DNA and load onto sterile macrocarrier/macrocarrier holder the day of experiment

Firing the Device

1. Plug in power cord from main unit to electrical outlet.

2. Power ON.

3. Sterilize chamber walls with 70% ethanol.

4. Load sterile rupture disk into sterile retaining cap. (Rupture can be immersed in iso-propanol when using)

5. Secure retaining cap to end of gas acceleration tube (inside, top of bombardment chamber) and tighten with torque wrench.

6. Load macrocarrier and stopping screen into microcarrier launch assembly.

7. Place microcarrier launch assembly and target cells in chamber and close door.

8. Evacuate chamber, hold vacuum at desired level (minimum 5 inches of mercury).

9. Bombard sample: Fire button continuously depressed until rupture disk bursts and helium pressure gauge drops to zero.

10. Release Fire button.

After the Bombardment

1. Release vacuum from chamber.

2. Target cells removed from chamber.

3. Unload macrocarrier and stopping screen from microcarrier launch assembly.

4. Unload spent rupture disk.

5. Remove helium pressure from the system (after all experiments completed for the day).

Choosing Bombardment Parameters (From Bio-Rad)

Cell Type	Vacuum (inches Hg)	Target Distance (cm)	Helium Pressure (psi)	Particle Size
Bacteria	29	6	1,100	M5 tungsten
Yeast	28	6	1,300	0.6 µm gold
Algae	29	6	1,300	0.6 µm gold
Plant
embryos	28	6	1,300	1.0 µm gold
callus or   cell cultures	28	9	1,100	1.0 µm gold
Subcellular organelles	28	6	1,300	0.6 µm gold
Animal
tissue cultures	15	3	1,100	1.6 µm gold
tissue sections	25	9	1,100	1.6 µm gold

We recommend these settings for bombardment of a variety of cell types. Many factors affect bombardment efficiency, but most users will find it sufficient to optimize the major variables individually, then test their interactions on a limited scale.