Helios Gene Gun System Instruction Manual Catalog Numbers 165-2431 and 165-2432 For Technical Service Call Your Local Bio-Rad Office or in the U.S.
Warranty and Regulatory Notices Warranty Statement This warranty may vary outside of the continental United States. Contact your local Bio-Rad office for the exact terms of your warranty. Bio-Rad Laboratories warrants to the customer that the Helios Gene Gun System (catalog number 165-2431 and 165-2432) will be free from defects in material and workmanship, and will meet all performance specifications for the period of one year from the date of shipment. This warranty covers all parts and labor.
Note: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part 15 of the FCC rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications.
Table of Contents Page Section 1 General Safety Information....................................................................1 1.1 1.2 1.3 1.4 Helios Gene Gun Safety........................................................................................1 Pressurized Helium and Nitrogen Safety..............................................................1 Power Safety..........................................................................................................1 Ear and Eye Protection.......
Section 1 General Safety Information Caution: In particle bombardment DNA-coated microparticles are accelerated to velocities in excess of 1,000 ft/sec in order to penetrate the cell membrane and through multiple layers of cells in tissues and organs. In the Helios Gene Gun, this accelerating force is supplied by a high pressure helium pulse. Numerous safety features have been designed into this instrument to protect both the user and bystanders.
Fig. 1. Location of the instrument serial number label on the Helios Gene Gun. 1.4 Ear and Eye Protection Caution: Expansion of gas from high pressure to low pressure produces a sound wave, the intensity of which is a function of the gas pressure. The intensity of the sound generated by discharging the Helios Gene Gun is ~108 decibels (db) at 400 psi; sustained noise levels of 85 db or brief noise levels of 110 db may lead to permanent hearing damage.
Table 1. Advantages of particle bombardment for in vitro and in vivo gene transfer.
Prior to transfection, the plasmid DNA must be attached to the gold particles. This is accomplished by precipitation of the DNA from solution in the presence of gold microcarriers and the polycation spermidine by the addition of CaCl2. The particles are then washed extensively with ethanol to remove the water and resuspended in ethanol. Using the Tubing Prep Station, the DNA/microcarrier solution is coated onto the inner wall of Gold-Coat tubing and dried. The tubing is then cut into 0.
Preparation of the gold/DNA tubes used in the Gene Gun requires an area approximately 1 m2 for the Tubing Prep Station, for manipulating the tubing, precipitating the DNA onto the gold, and processing the tubing into cartridges. Additionally, the Tubing Prep Station requires an electrical outlet and a tank of pressurized nitrogen for evaporating the ethanol from the DNA-coated gold particles from the inner surface of the tubing.
Battery One battery is provided with the Helios Gene Gun System. Under normal use, it should provide approximately 1,000 discharges. For maximum life, only alkaline batteries should be used. Laboratory Equipment The following materials should be available before beginning any work with the Helios system. Ultrasonic cleaner (e.g., Fisher FS3, Branson 1210) Vortex mixer Analytical balance Microfuge Peristaltic pump capable of pumping 5–8 ml/min (e.g. Bio-Rad Econo Pump, catalog number 731-8140) 1.
Section 3 Product Description 3.1 Packing List The Helios Gene Gun System (see Figures 3 and 4) is shipped with the following components. If items are missing or damaged, contact your local Bio-Rad office.
Fig. 3. Major components used for sample delivery with the Helios Gene Gun. Fig. 4. Components of the Tubing Prep Station. 3.2 Identification of System Components and Controls Helios Gene Gun The locations front and back refer to the barrel end and LED display end of the Helios Gene Gun, respectively. The locations left and right refer to the left and right sides of the Gene Gun from the viewpoint of the user holding the device.
Fig. 5. Components and controls on the Helios Gene Gun. Gene Gun Controls Cylinder Lock Description Controls movement of the barrel pin. The cylinder lock is spring-loaded; its natural position is in the backward (locked) position so the barrel pin is inserted in the hole in the cartridge holder; this keeps the cartridge holder in its proper position for firing. Moving the cylinder lock forward disengages the barrel pin from the cartridge holder to permit removing the cartridge holder from the gun.
Push Bar A metal bar that ratchets the cartridge holder from one position to the next when the Cylinder Advance Lever is pressed. Move this bar to the left (outward) prior to inserting a cartridge holder to provide additional room for maneuvering the cartridge holder. LED Display An 11 light display.The display is normally off; inserting a cartridge holder in the Gene Gun and advancing to position 1 activates the display. The left-most 7 LEDs act to indicate charging and ready status of the gun.
Fig. 6. Components and controls on the Tubing Prep Station, fully assembled. Fig. 7. The Tubing Cutter. Cartridge Extractor Tool (see Figure 8) A 12-prong tool for removal of tubes from the cartridge holder. One prong is longer than the others so it can be easily inserted into one of the bores of the cartridge holder to orient the remaining 11 prongs.
Fig. 8. Cartridge Holder and Cartridge Extractor Tool. Section 4 Setting up the Helios Gene Gun System 4.1 Inserting the Battery into the Helios Gene Gun The electrical system of the Helios Gene Gun is powered by a 9 volt battery. Under normal use, this should provide sufficient energy for 1,000 shots. The battery compartment is in the base of the handle near the attachment fitting for the helium hose (Figure 9).
Fig. 9. Battery compartment. The battery compartment is located at the base of the handle of the Gene Gun next to the connection for the helium hose and is protected by a battery cover that slides forward. The battery is inserted with the positive terminal (the smaller of the two terminals) facing forward. 4.2 Connecting the Helios Gene Gun to a Helium Source Refer to Section 1, Safety Information, and Section 3.2, Identification of System Components and Controls, prior to system installation.
Attaching the Helios Gene Gun to the Helium Regulator Components needed Helium regulator attached to a helium cylinder Helium hose assembly Helios Gene Gun Procedure 1. Insert the stem of the Swagelok Quick-Connect fitting on the helium hose into the opening in the body of the Swagelok Quick-Connect fitting on the helium regulator and push until it clicks. The helium hose will be locked into the helium regulator (see Figure 10).
Assembly of the Tubing Prep Station and Syringes Components needed Tubing Prep Station, base Tubing Prep Station, tubing support cylinder Tubing Prep Station, power cord O-rings, for tubing support cylinder Syringe adapter tubing (silicone, 5 ft, 0.104" ID, 0.192" OD) 10 cc syringes (2) 10 cc syringe sleeve 1/8" barb-to-male Luer fittings (2) 1/8" barb-to-female Luer fittings (2) Scissors, user supplied Fig. 11. Components and controls on the Tubing Prep Station, fully assembled. Procedure 1.
4. Cut a 12–13" piece of syringe adapter tubing; attach one end of the tubing to the barb of a 1/8" barb to female Luer fitting; attach the female Luer to a 10 cc syringe. This syringe and tubing will be used to load the DNA/microcarrier suspension into the Gold-Coat tubing (Section 5.1). 5. (Optional—if peristaltic pump not used) Cut a 16-18" piece of syringe adapter tubing; attach one end of the tubing to the barb of a 1/8" barb-to-female Luer fitting; attach the female Luer to a 10 cc syringe.
4. The nitrogen regulator should be turned on and adjusted to the correct pressure prior to connecting the nitrogen line to the Tubing Prep Station. Close the nitrogen pressure regulator by turning the regulator adjustment screw counterclockwise until the adjusting spring pressure is released and the screw moves without resistance. Release nitrogen into the pressure regulator by carefully and slowly opening the cylinder valve on the nitrogen tank.
5.2 Preparation of System Components Prior to Bombardment Calculating the Amounts of Gold and Plasmid Required Prior to precipitating DNA onto the gold particles and loading them into the Gold-Coat tubing, it is necessary to calculate the amount of DNA and gold required for each transformation. Points to consider in making these calculations are presented below. The amount of DNA loaded per mg of microcarriers is referred to as the DNA Loading Ratio (DLR). Typical DLRs range between 1 and 5 µg DNA/mg gold.
Table 2. Microcarriers and DNA Required for Various Microcarrier Loading Quantities (MLQ) and DNA Loading Ratios (DLR)1 Calculated Particle Delivery Conditions MLQ DLR (µg/ (µg/ (mg/shot) mg gold) shot) 0.5 2 1 0.125 8 1 0.25 4 1 0.75 1.33 1 1.0 1 1 0.5 0.002 0.001 0.5 0.02 0.01 0.5 0.2 0.1 0.5 10 5 Materials Required for Selected MLQ’s and DLR’s Final Gold DNA volume Tubing (mg) (µg) (ml)2 (total in)3 50 100 6.0 50 12.5 100 6.0 50 25 100 6.0 50 75 100 6.0 50 100 100 6.0 50 50 0.1 6.0 50 50 1 6.
Procedure Time considerations: preparation of the DNA/gold suspension requires approximately 30 min. Several samples may be prepared simultaneously without a significant increase in time. 1. Prepare a stock solution of 20 mg/ml PVP in ethanol in a small screw-cap container. Dilute this solution with ethanol to prepare PVP solutions at the desired concentration (generally 0.01–0.1 mg/ml); prepare 3.
Loading the DNA/Microcarrier Suspension into Gold-Coat Tubing Using the Tubing Prep Station Materials Supplied Tubing Prep Station (see Section 4.3) Gold-Coat tubing To be Supplied by User Microcarrier/DNA suspension(s) from Section 5.1, Precipitation of DNA onto Microcarriers, at room temperature Ultrasonic cleaner Vortexer 100% ethanol Peristaltic pump Minute timer Nitrogen tank (see Section 2.4) Nitrogen regulator (see Section 2.
6. Remove the Gold-Coat tubing from the Tubing Prep Station. Turn off the flow of nitrogen to the Tubing Prep Station using the knob on the flowmeter. 7. From the dried Gold-Coat tubing cut a 29–30" (~75 cm) length of tubing for each 3 ml sample of Microcarrier/DNA suspension. (Note: Cutting the tubing with a scissors may distort the shape of the end; the tubing may be easier to insert into the tubing support cylinder if the end is subsequently cut in the tubing cutter.
Procedure 1. Examine the coated the Gold-Coat tubing to verify that the microcarriers are evenly distributed over the length of the tubing. Ideally, the gold should be spread uniformly over the entire inside surface of the tubing; however, while drying, it may polarize to one side of the tubing. As long as there are no clumps or bare sections, the tubing can be used for cartridges. 2. Using scissors, cut off and discard sparsely and unevenly coated tubing from one of the ends.
Fig. 12. Positioning the cylinder lock and the push bar in preparation for loading a cartridge holder into the Gene Gun. The cylinder lock has been pushed forward and into the slot on the right to move the locking pin away from the opening occupied by the cartridge holder. Likewise, the push bar has been pulled to the left to increase the space for inserting the cartridge holder. Fig. 13. Inserting a cartridge holder into the Gene Gun.
c. Pull back and hold the cylinder advance lever to retract the inner barrel sleeve into the gun barrel (Figure 13). d. Place the empty cartridge holder into the Gene Gun with the position 12 label facing up and the numbered side of the cartridge holder facing the exit nozzle of the barrel (Figure 12). e.
Fig. 15. LED display of the Helios Gene Gun. Using a pressurized system, once the cartridge holder of the Gene Gun is correctly inserted and engaged in postion 1, the five CHARGING lights will be sequentially illuminated. Once the unit is fully charged (~5 sec), the CHARGED light will flash. If the safety interlock is then pressed, the ARMED lights will alternately flash.
Notes: (1) The firing trigger is functional only while the safety interlock switch is pushed in. The safety interlock switch activates the trigger button for 30 sec; if the Gene Gun is not fired within the 30 sec period, the safety interlock switch must be released, then pressed again to reactivate it. (2) After firing the gun, there is a 5 sec delay before the gun may be fired again. (3) Advance the cartridge holder by squeezing the cylinder advance lever between each shot. Fig. 16.
Fig. 17. Loading cartridges into the cartridge holder. The numbers located on the outer rim indicates sample number when delivered by the Gene Gun. DNA delivery to target tissue (see Section 6 for suggestions on preparing mammalian target cells) 1. Touch the target area with the spacer so that the spacer is flush and the Gene Gun is perpendicular to the target surface. Activate the safety interlock switch and press the trigger button to deliver the DNA/microcarriers to the target. 2.
2. Turn the regulator valve counterclockwise until both the high and low pressure gauges on the helium regulator register 0 psi. Several increase/decrease adjustments on the regulator may be necessary. Listen for venting to ensure complete depressurization. The system is now depressurized and can be safely disassembled. 3. Disconnect the helium hose from the regulator by pulling the sleeve on the Swagelok Quick-Connect coupling toward the helium hose and pulling the fittings apart. 4.
7. Immediately prior to DNA delivery, aspirate the media from the dish. 8. Hold the dish perpendicular to the spacer and touch the end of the plastic spacer (if the spacer is sterile), or as close as possible to the target, and discharge the Gene Gun (Figure 18). 9. Add 1.5–2 ml of media to the plate and return to the incubator. 6.2 In vitro Delivery to Suspension Cultures Method 1 1. Prepare the Helios Gene Gun for operation as described in Section 5.3. 2.
5. Incubate 30 min to 4 hr under the appropriate conditions. 6. Prepare the Helios Gene Gun for operation as described in Section 5.3. 7. Immediately prior to DNA delivery, aspirate the media from the dish. 8. Hold the dish perpendicular to the spacer and touch the end of the plastic spacer (if the spacer is sterile), or as close as possible to the target, and discharge the Gene Gun (Figure 18). 9. Add 1.5–2 ml of media to the plate and return to the incubator. 6.
Section 7 Optimization of Gene Gun Parameters 7.1 Overview The flexibility of the particle delivery system allows fine-tuning of experimental parameters; however, it is necessary for each laboratory to determine the optimal parameters for their particular instrument and in vivo or cell culture system. Any quantitative assay may be used to determine the optimum combination of critical parameters for the particular biological system under investigation.
hGH activity (ng/ml) 80 60 40 -6 20 0 100 200 Helium pressure (psi) 400 Fig. 20. Luciferase expression in mouse skin transfected in vivo using the Helios Gene Gun. Skin homogenates were assayed 24 hours post-transfection. Luciferase relative light units -8x 10 Luciferase activity (relative light units x 10-8) As illustrated in Figure 21, the optimum MLQ is determined by adjusting the concentration of the DNA/microcarrier suspension loaded into the Gold-Coat tubing.
Another variable parameter, DNA loading rate (DLR), is determined by varying the concentration of DNA precipitated on to the gold particles. Table 3 shows DNA dosage results for transfection of CHO cells using the method described in Section 6.2. Secretion of murine granulocyte macrophage colony stimulating factor (mGM-CSF) was greater at higher DNA dosages. Similar results have been found for several in vivo and in vitro systems, with maximal expression observed for 1–5 µg DNA/mg gold particles.
Table 4. Suggested starting conditions for in vitro transformation of tissue culture cells using the Helios Gene Gun. Parameter Helium pressure: Conditions 50–200 psi PVP: 0–0.015 mg/ml MLQ: 0.125–0.5 mg/tube Microcarriers: 1.0 or 1.6 µ gold DLR: 0.5–2.5 µg DNA/mg gold (0.06–1.25 µg DNA/cartridge) 7.3 Parameters for in vivo Delivery The following are suggested starting conditions for optimizing DNA delivery to the skin of various species.
1. Rewash remaining microcarrier prep 2–3 times using a fresh bottle of 100% ethanol. 2. Flush Gold-Coat tubing with nitrogen gas for 15 min prior to loading with DNA/gold microcarrier solution. 3. If using PVP, make a new stock of PVP in ethanol using a fresh bottle of alcohol. 4. Resuspend gold/DNA pellet prior to first ethanol wash. 5. After drawing off the ethanol, quickly turn the Gold-Coat tubing with your fingers prior to rotating. 8.3 Helios Gene Gun Operation Problem LED display not lit.
Possible solutions 1. Make sure DNA is resuspended in TE or distilled water, not saline. 2. Check that DNA has been precipitated onto the microcarriers (see Section 10.7). Problem Attached cells are lifting off the center of the plate after delivery of gold particles, or there is a dead zone in the center of the target. Possible solutions 1. Lower the helium pressure and/or lower the microcarrier loading rate. Problem Contamination of the cell culture.
(polyvinylpyrrolidone, MW 360,000), 0.5 g, Cartridge Collection/ Storage Vials, 5, Dessicant Pellets, 5, Gold-Coat Tubing, 50 ft. 165-2432 Helios Gene Gun System, 220/240 V, as above for 165-2431, except 165-2420 Tubing Prep Station, 220/240 V, 1 9.
Environmental Operating Storage 50 °F ( 10 °C) to 90 °F (32 °C) temp. 30–80 % humidity 32 °F (0 °C) to 140 °F (60 °C) temp. 10–90 % humidity Tubing Prep Station Specifications Physical Dimensions Weight Construction Approximately 33.5” x 4” 11.2 lbs Aluminum and Acrylic Electrical Maximum Current Voltage Input Input Frequency 62 mA/125 mA peak 220–240 Vac/100–120 Vac 50/60 Hz Functional Input Pressure Relief Pressure Regulator Adjustment Speed Tubing Fill 30 psi N2 30 psi ± 1.
2. To measured gold, add aqueous solution of mRNA preparation. The ratios of RNA to gold used are similar to those used for DNA (1–15 µg RNA/mg particles). 3. Add 1/10 volume of 5 M ammonium acetate and 2 volumes of 2-propanol, mix by vortexing. Place the microfuge tube at -20 °C for 1 hour. 4. Proceed with Section 5.2, steps 9–12. 10.2 Replacing the O-rings and Barrel Liner on the Helios Gene Gun Fig. 22. Location of barrel liner and O-rings in the Gene Gun.
To replace the O-ring on the end of the barrel liner: 1. Remove the barrel liner. 2. Remove the old O-ring from the barrel liner. 3. Place a new O-ring in the groove and press it in gently. Do not use grease or adhesive. To replace the O-ring around the exit port of the main valve: 1. Using a pin or syringe needle, poke the O-ring from the edge to pop it out. Be careful not to scratch the surface of the valve. 2. Place a new O-ring in the groove and press it in gently. Do not use grease or adhesive. 10.
10.4 Replacing the razor blade on the Tubing Cutter and disassembly of the unit The cutting edge of the Tubing Cutter is a standard, single edge razor blade. Because cutting is done only on one end of the blade at once, it can be rotated after the first end becomes dull. The first sign of a dull blade will be incomplete cutting of two adjacent cartridges. Front refers to the side of the Tubing Cutter with the tubing channels.
3. Raise the arm up and away from the base. 4. The Tubing Cutter may be cleaned with soap and water and /or with 70% or 100% ethanol. Do not autoclave the Tubing Cutter. 5. Assembly is the reverse of disassembly. With the spring in place, lay the cutting arm, correctly oriented, on top of the base. With the index and middle fingers of your left hand, press down on the left side of the arm to compress the spring about 3/16”.
Discharge into Parafilm Materials Parafilm laboratory film (American Can Company) Glass plate Procedure 1. Cut a piece of Parafilm from the roll; a 1" length is required for each cartridge to be assayed. 2. Smooth and press the Parafilm laboratory film (waxy surface down) onto the glass plate. Fold the edges around the sides then peal away the protective paper. 3. Connect the helium regulator, helium hose, and Helios Gene Gun as described in Section 4.2. 4. Pressurize the system as described in Section 5.3.
Fig. 26. Representative cross-section showing penetration of gold particles into water agar after discharge with a helium pulse. 10.7 Quantitation of DNA in Cartridges This procedure can be used for estimating the amount of DNA in cartridges when 0.5 µg or more of DNA has been loaded per tube. Materials 10 mM Tris, pH 8.0, 1 mM EDTA Ultrasonic cleaner Vortexer Microfuge 1,000 µl micropipettor and tips UV spectrophotometer Quartz cuvettes Procedure 1. Place five 0.
10.8 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 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). 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). Armaleo, D., Ye, G. N, Shark, K. B., Sanford, J. C. and Johnston, S. A., Curr. Genet. 17, 97-103 (1990).
10.9 Quick Guide to Operation Before the Bombardment 1. Coat microcarriers with DNA, load into tubes, and prepare cartridges prior to day of experiment. 2. Check helium supply (50 psi in excess of desired rupture pressure). 3. Clean and/or sterilize the Gene Gun, tube holders, and barrel liner as appropriate. 4. Connect the Gene Gun to a helium source. 5.
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