BIO RAD Helios Gene Gun System Instruction Manual
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BIO RAD Helios Gene Gun System Instruction Manual

BIO RAD Helios Gene Gun System Instruction Manual

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Helios Gene Gun System
Instruction Manual
Catalog Number # 1652431
1652432

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Summary of Contents for BIO RAD Helios Gene Gun System

  • Page 1 Helios Gene Gun System Instruction Manual Catalog Number # 1652431 1652432...
  • Page 2 Warranty and Regulatory Notices Warranty Statement The Helios Gene Gun System and associated accessories are covered by a standard Bio-Rad warranty. Contactyour local Bio-Rad Laboratories office for details of the warranty. Regulatory Notices Important: This Bio-Rad instrument is designed and certified to meet EN/IEC 61010-1:2010 (3rd Edition), EN61326- 1:2013 Class A, and EN50581:2012 requirements, which are internationally accepted electromagnetic compliance and electrical safety standards.

  • Page 3: Table Of Contents

    Particle Delivery Using the Helios Gene Gun System ....... . .
  • Page 4 IV | Helios Gene Gun System...

  • Page 5: General Safety Information

    Please follow the instructions provided with the helium tank from the supplier and those that are applicable for your institution (see your Site Safety Officer). Bio-Rad has supplied tubing, fittings, a control valve, and a pressure regulator capable of safely handling the high-pressure helium gas used in the Helios Gene Gun.

  • Page 6: Power Safety

    Helios Gene Gun. Earmuffs or ear plugs provide equivalent protection against hearing damage. Refer to Requirements for System Operation in Section 2 for suggestions on ear protection. Eye protection should always be worn when working with gases under high pressure. 2 | Helios Gene Gun System...

  • Page 7: Introduction To Particle Delivery

    The Helios Gene Gun The Helios Gene Gun is the second instrument in Bio-Rad’s particle delivery product line. In contrast to the PDS-1000/He System, in which the overall size of the target to be transformed is limited by the size of the chamber, and the target tissue is subjected to a vacuum during bombardment, the Helios Gene Gun requires no vacuum and any target accessible to the barrel can be transformed.

  • Page 8: Operating Principle Of The Helios Gene Gun System

    Operating Principle of the Helios Gene Gun System The Helios Gene Gun System consists of all of the components needed to prepare DNA-coated microcarriers, coat the DNA-microcarrier suspension onto the inner surface of the GoldCoat Tubing, cut the tubing into cartridges, which are used in the Helios Gene Gun, and finally propel the microcarriers and their associated DNA into cells.

  • Page 9: Requirements For System Operation

    Prep Station and 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. Helios Gene Gun System | 5...

  • Page 10: User Supplied Components

    (1) a self-venting valve that permits decreasing the pressure in the Helios Gene Gun System in the event of battery failure or when it is necessary to reduce the pressure during an experiment; (2) an over-pressure relief valve that prevents the helium pressure in the Helios Gene Gun System from being adjusted above 700 psi ±...
  • Page 11 Section 2 Introduction to Particle Delivery Battery One battery is provided with the Helios Gene Gun System. Under normal use, it should provide approximately 1,000 discharges. For maximum life, use only alkaline batteries. Laboratory Equipment The following materials should be available before beginning any work with the Helios System.

  • Page 12: Product Description

    Bio-Rad’s Quantum Prep Plasmid Prep Kits. See Section 9, Product Information, on page 41. Section 3 Product Description 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. Helios Gene Gun Kit Instruction manual ■...
  • Page 13 5 cartridge collection/storage vials ■ Note: If any of the system components (Helios Gene Gun, Tubing Prep Station, tubing cutter, helium regulator, or helium hose) are dropped, check them for proper operation before use. Helios Gene Gun System | 9...

  • Page 14: Identification Of System Components And Controls

    (see Figure 5). LED display FRONT BACK Safety interlock button Push bar Trigger button Cylinder advance lever Battery access cover Fig. 5. Components and controls on the Helios Gene Gun. 10 | Helios Gene Gun System...
  • Page 15 O-ring into which the tubing must be inserted. The tubing support cylinder can be removed by pushing it to the right/left to compress the spring that holds it in position. Helios Gene Gun System | 11...
  • Page 16 The support for the arm. It positions a storage vial under the tubing channels so that the cut tubing pieces fall into the vial. Tubing channels Razor blade Base Fig. 7. The tubing cutter. 12 | Helios Gene Gun System...
  • Page 17 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. Cartridge holder Cartridge extractor tool Fig. 8. Cartridge holder and cartridge extractor tool. Helios Gene Gun System | 13...

  • Page 18: Inserting The Battery Into The Helios Gene Gun

    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. 14 | Helios Gene Gun System...

  • Page 19: Connecting The Helios Gene Gun To A Helium Source

    U.S. is compatible with the fitting supplied on the pressure regulator with the Helios Gene Gun System (CGA 580, female fitting). Outside the U.S., contact your local Bio-Rad office for information regarding the proper cylinder/regulator fitting in your area.

  • Page 20: Setting Up The Tubing Prep Station

    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', 0.104" ID, 0.192" OD) ■ 16 | Helios Gene Gun System...
  • Page 21 10 cc syringe. This syringe and tubing will be used to load the DNA/microcarrier suspension into the GoldCoat Tubing (Guide to Operation in Section 5). Helios Gene Gun System | 17...
  • Page 22 Pressure regulator for nitrogen cylinder, user supplied (see Requirements for System Operation, in ■ Section 2). A regulator is available from Bio-Rad (#1652425), which is ready for use in the Helios Gene Gun System Nitrogen tank of grade 4.8 or higher (99.998% pure); maximum pressure of 2,600 psi, user supplied ■...

  • Page 23: Operation Of The Helios Gene Gun System

    Section 5 Operation of the Helios Gene Gun System Procedure 1. Determine the length of nitrogen hose needed to connect the Tubing Prep Station to the the nitrogen regulator and cut it if necessary. 2. Connect the 3/16" barb-to-male luer fitting to one end of the nitrogen hose.

  • Page 24: Preparation Of System Components Prior To Bombardment

    Section 5 Operation of the Helios Gene Gun System After the Bombardment 1. Remove cartridge holder from gene gun. 2. Remove cartridges from cartridge holder. 3. Turn off the helium pressure to the system. 4. Turn the regulator value counterclockwise to depressurize the system.
  • Page 25 Section 5 Operation of the Helios Gene Gun System Table 2. Microcarriers and DNA required for various microcarrier loading quantities (MLQ) and DNA loading ratios (DLR).* Calculated Particle Delivery Conditions Materials Required for Selected MLQs and DLRs MLQ, DLR, DLR,...
  • Page 26 Section 5 Operation of the Helios Gene Gun System − Purified plasmid DNA resuspended in distilled water or 10 mM Tris (pH 8.0), 1 mM EDTA − Ultrasonic cleaner (for example, Fisher FS3, Branson 1210) − Analytical balance capable of weighing microgram quantities −...
  • Page 27 Section 5 Operation of the Helios Gene Gun System 12. The suspension is now ready for tube preparation. Alternatively, the DNA/microcarrier suspensions can be stored for up to 2 months at –20°C. Prior to freezing, tighten the cap securely and put Parafilm Laboratory Film around the cap of the tube.
  • Page 28 Section 5 Operation of the Helios Gene Gun System Tefzel Tubing at 0.5–1.0"/sec. Practice removing liquid at this rate from the Tefzel Tubing by cutting a ~30" piece of tubing, loading it with ~3.0 ml of ethanol (~24" (~60 cm) of tubing), inserting it into the Tubing Prep Station, and removing the liquid at 0.5–1.0"/sec.
  • Page 29 Section 5 Operation of the Helios Gene Gun System 15. Turn the motor on the Tubing Prep Station to OFF (O). Turn the nitrogen off by closing the valve on the flowmeter. Remove the tubing from the tubing support cylinder.

  • Page 30: Particle Delivery Using The Helios Gene Gun System

    Section 5 Operation of the Helios Gene Gun System Particle Delivery Using the Helios Gene Gun This section describes the procedure for preparing the Helios Gene Gun for firing, discharging the device, loading cartridges into the cartridge holder, and delivering DNA to target cells. (Warning: Use the Helios Gene Gun in a well ventilated area.)
  • Page 31 Section 5 Operation of the Helios Gene Gun System Fig. 13. Inserting a cartridge holder into the gene gun. Pulling the cylinder advance lever in moves the barrel forward to increase the space for inserting the cartridge holder behind the barrel liner. The cartridge holder is inserted with the labeled position number 12 (visible at the top) in line with the center of the gene gun (see also Figure 12).
  • Page 32 Section 5 Operation of the Helios Gene Gun System Fig. 14. Correct assembly of the cartridge holder in the gene gun in preparation for discharge. Releasing the cylinder advance lever moves the barrel liner backward, bringing the O-ring on the back of the liner in contact with the cartridge holder. Releasing the cylinder lock inserts the locking pin into the center hole in the cartridge holder.
  • Page 33 Section 5 Operation of the Helios Gene Gun System i. Push in and release the cylinder advance lever to ratchet the cartridge holder one position, bringing the first cartridge into firing position. The number 12 should be seen at the top point of the cartridge holder.
  • Page 34 Section 5 Operation of the Helios Gene Gun System a. For right handed users (see Figure 16): Engage the safety interlock switch by pushing in with the thumb. Push the firing trigger with the index finger to discharge the cartridge.

  • Page 35: Removing Used Cartridges, Depressurization, And Shutdown

    Section 5 Operation of the Helios Gene Gun System 1. Place the cartridge holder on a flat surface with the numbered edge facing up. 2. Starting with position 1, load up to 12 cartridges into the cartridge holder (Figure 17). We recommend loading only one type of DNA per cylinder to avoid possible confusion.

  • Page 36: Preparation Of Mammalian Cell Targets

    4. Allow the plates to incubate undisturbed for 30 min to permit the cells to attach. 5. Gently add 1 ml of media to each plate and place in the incubator. Incubate the cells for at least 4 hr. 32 | Helios Gene Gun System...

  • Page 37: In Vitro Delivery To Suspension Cultures

    1.5 cm circle in the center of a 35 mm dish. It is useful to draw a 1.5 cm diameter circle on an index card for use as a template under the culture dish. Helios Gene Gun System | 33...
  • Page 38 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. 34 | Helios Gene Gun System...

  • Page 39: In Vivo Delivery To Epidermis

    Some animals may show transient erythema or inflammation at the treatment site.) Fig. 19. In vivo bombardment of murine epidermis (photo courtesy of Auragen, Inc.). Helios Gene Gun System | 35...

  • Page 40: Optimization Of Gene Gun Parameters

    200 psi. This pressure resulted in adequate penetration of the particles without excessive tissue damage, and deposition of the majority of the particles in the epithelial layer rather than the relatively acellular underlying dermal tissue. 36 | Helios Gene Gun System...
  • Page 41 Fig. 21. Effect of the amount of accelerated gold particles on in vitro expression levels. Suspension cultures of CHO cells were bombarded as described in Section 6.2 using the Accell Gene Gun. After 18 hours, the CHO cells that had reattached to the tissue culture plate were lysed and assayed for luciferase expression. Helios Gene Gun System | 37...

  • Page 42: Parameters For In Vitro Delivery

    Table 4. Suggested starting conditions for in vivo transformation of tissue culture cells using the Helios Gene Gun. Parameter Conditions Helium pressure 50–200 psi 0–0.015 mg/ml 0.125–0.5 mg/tube Microcarriers 1.0 or 1.6 µ gold 0.5–2.5 µg DNA/mg gold (0.06–1.25 µg DNA/cartridge) 38 | Helios Gene Gun System...

  • Page 43: Parameters For In Vivo Delivery

    If using PVP, make a new stock of PVP in ethanol using a fresh bottle of alcohol. Resuspend gold/DNA pellet prior to first ethanol wash. After drawing off the ethanol, quickly turn the GoldCoat Tubing with your fingers prior to rotating. Helios Gene Gun System | 39...

  • Page 44: Helios Gene Gun Operation

    Open the helium regulator quickly one or two turns and pressurize the system to >100 psi. The pressure can then be readjusted to another setting. If continuous helium discharge is observed after this, contact Bio-Rad. In Vitro and In Vivo Targeting Problem Gold does not penetrate skin or organ.

  • Page 45: Product Information

    The catalog numbers for the two Helios Gene Gun Systems (100/120 V and 220/240 V) and component descriptions are as follows: Catalog # Description 1652431 Helios Gene Gun System, 100/120 V includes 1652411 Helios Gene Gun Kit, that includes Gene Gun Unit, 1 1652416 O-rings, Helios Gene Gun, 5...

  • Page 46: Spare Parts

    700 ± 35 psi at regulator assembly Regulator adjustment 800 psi limit maximum Discharges 12 per cylinder revolution Indexing Manual Environmental Operating 50–90°F (10–32°C) temp 30–80% humidity Storage 32–140°F (0–60°C) temp 10–90 % humidity 42 | Helios Gene Gun System...

  • Page 47: Tubing Prep Station Specifications

    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. Helios Gene Gun System | 43...

  • Page 48: Replacing The O-Rings And Barrel Liner On The Helios Gene Gun

    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. 44 | Helios Gene Gun System...

  • Page 49: Replacing The O-Ring On The Tubing Prep Station

    2. Move the lock block to the right to free the razor blade retaining screw on the right side. 3. Lift up on the right side of the razor blade until it can be lifted free of the tubing cutter. Helios Gene Gun System | 45...
  • Page 50 46 | Helios Gene Gun System...

  • Page 51: Cleaning And Sterilizing The Helios Gene Gun

    GoldCoat Tubing. Fig. 25. Representative bombardment showing penetration of 1.6 µm gold particles into Parafilm Laboratory Film at (left to right) 100, 200, 300, 400, 500, and 600 psi using the Helios Gene Gun. Helios Gene Gun System | 47...
  • Page 52 2. A band of microcarriers will be visible in the agar with increasing particle sizes at increased depths. The researcher will be able to determine areas of high, medium, and low microcarrier density in each slice (Figure 26). 48 | Helios Gene Gun System...
  • Page 53 For this reason, in order to obtain a truly representative microcarrier distribution cross section, be careful to examine the center and full length of your target site. Fig. 26. Representative cross-section showing penetration of gold particles into water agar after discharge with a helium pulse. Helios Gene Gun System | 49...

  • Page 54: Quantification Of Dna In Cartridges

    TE blank. If 100% of the DNA were precipitated onto the microcarriers, the A readings should be as follows: Plasmid/Cartridge, µg 4. DNA sample may also be analyzed by restriction enzyme digestion and analysis by agarose gel electrophoresis. 50 | Helios Gene Gun System...

  • Page 55: References

    The instruments used in these experiments vary. Use the above information as a starting point when optimizing bombardment conditions with the Helios Gene Gun. Lower helium pressures should be tested early in the optimization process. Helios Gene Gun System | 51...

  • Page 56: Quick Guide To Operation

    4. Turn the regulator value counterclockwise to de-ressurize the system. 5. Disconnect the helium hose and gene gun. Go to bio-rad.com/Helios/training for additional Helios Gene Gun training and overview information, including instrument setup, microcarrier and cartridge preparation, and instrument operation. 52 | Helios Gene Gun System...
  • Page 60 Life Science Web site bio-rad.com USA 1 800 424 6723 Australia 61 2 9914 2800 Austria 43 01 877 89019 Belgium 32 03 710 53 00 Brazil 55 11 3065 7550 Canada 1 905 364 3435 China 86 21 6169 8500 Czech Republic 36 01 459 6192 Denmark 45 04 452 10 00...

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