Michael Nonet, Department of Anatomy and Neurobiology, Washington University School of Medicine, Saint Louis, MO 63110 nonetm@pcg.wustl.edu 314-747-1176

(Click on picture for larger labeled image)

Click on pictures for larger labeled images

Gun

trigger wiring

solenoid valve

small parts

NOTE: Helium tank and regulator are not shown

The gun is essentially a solenoid valve triggered by a relay switch to fire for 50 milliseconds. The gun is a modification of a gene gun built in David Kirk's lab at Washington University to transform Volvox. It does not use a vacuum. It has been used successfully by my lab to create C. elegans transgenes, and by Paul Bridgman's lab to transform cultured neurons. The cost depends on whether you have a helium regulator or not. All together, including the regulator and helium tank, it can be purchased for around $500.00. Prices reflect the discounts that Washington University gets from various companies, but that shouldn't change prices too much.

The gun

The gun consists of a solenoid valve with 1/8 inch NPT female fittings. On the input side the solenoid is attached to tubing to connect to the regulator, on the other side it is attached to a Swinnex filter holder which acts as the support to load the gold or tungsten particles you wish to fire. The cheap way to connect to the Swinnex filter to the solenoid is to shave of the threading on the Swinnex filter. It then fits tightly into the Parker O-ring fitting. A more elegant and expensive approach is to purchase a 1/8" NPTM to M Luer locking adapter from Millipore (see parts list). For C. elegans applications, the Swinnex filter bottom is cut so that the gold spread is more diffuse. For a more condensed spray of gold, the tip can be left uncut. I originally purchased a 300 psi solenoid valve, but I am now firing at 400 psi using the 300 psi valve. It holds the pressure now, but may not forever. One could purchase a solenoid valve that will hold 750 psi for essentially the same price (see options in parts list).

DNA-covered gold particles are loaded onto the Millipore filter holder male end in 5 to 10 ml of ethanol. The gold spreads relatively evenly onto the plastic support using this volume. The ethanol doesn't cause any harm andmost of it probably evaporates before hitting the cells during firing. The filter is screwed into the female end, and we fire from about 7 cm using aa 300- 400 psi burst for 50 ms. I am securing the solenoid using a mounting clamp attached to a ring stand so I can adjust the height easily.

The trigger mechanism

The solenoid is wired to a trigger that consists of a momentary swich which fires a one-shot relay swich. The wiring can be figured out from looking at the photograph. Note that the 120 V AC current powers both the relay and the solenoid. The relay can be set to fire by 10 millisecond intervals from 0.01 to 10.00 seconds. The solenoid won't fire reliably unless you use at least 30 to40 milliseconds fireing time. We usually use 50 milliseconds (005 on the relay). The relay should be set to one shot mode. I mounted the trigger mechanism into an old Starsted blue tip box, but I am sure it can be done more elegantly. The momentary switch is overkill, but originally I was trying to fire the solenoid using the momentary switch without the relay. This works, but it is hard to get a reproducible length helium burst that way. The only purpose of the relay it to have consistency in the helium burst from one shot to another. The switch doesn't need to be a momentary switch, but if you use a regular switch, you'll need to turn the switch back off each time, before you can fire again.

The helium regulator

I purchased a regulator that could provide up to500 psi delivery pressure and purchased a moderately priced one so that I could use it for other applications. If you plan to shoot vertebrate cells you probably won't need to go above 150 psi. Be careful when you purchase helium. You want high purity helium to insure that that there won't be any mold spores in the tank. I connected up the helium tank to the solenoid valve using high pressure Teflon tubing because I had some around. I listed a nylon tubing source because I am not sure if one can purchase small lengths of the Telfon tubing. It gets expensive in a 25 or 50 ft lengths. There are many ways to connect this up and what kind of tubing you use will depend on the pressure you want to use.

Distribution of gold using this apparatus

We analyzed the distibution of particles using our set up when shot onto a 60 mm plate from 7 cm at 300 psi. Click on numbers on the left to see an image of gold distribution at that point on the plate. On the right click to see distribution at various depths in the agar. As you can see, we get a nice broad distribution.

Please send comments regarding this apparatus to nonetm@pcg.wustl.edu

Parts List

Airgas www.airgas.com

Fisher www.fishersci.com

Grainger www.grainger.com

Millipore www.millipore.com

Radioshack www.radioshack.com

United States Plastic Corporation www.usplastic.com