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BIO RAD Helios Gene Gun System, Инструкция по эксплуатации

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Section 10 Appendices 

Helios Gene Gun System  |  49

3.  To determine the depth of penetration, align the 0 of an eyepiece micrometer at the upper surface of 

the agar and measure the distance to the arbitrary floor of the microcarrier distribution. This value is the 
microcarrier depth.

The distribution pattern of the microcarriers is bell-curve shaped with respect to depth and area. The 
highest microcarrier density is concentrated at the center of the shot and at the surface of the target site. 
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.

Содержание Helios Gene Gun System

Страница 1: ...Helios Gene Gun System Instruction Manual Catalog Number 1652431 1652432...

Страница 2: ...be modified or altered in any way Alteration of this instrument will void the manufacturer s warranty void the regulatory certifications and create a potential safety hazard Note This equipment has b...

Страница 3: ...epressurization and Shutdown 31 Section 6 Preparation of Mammalian Cell Targets 32 In Vitro Delivery to Adherent Cells 32 In Vitro Delivery to Suspension Cultures 33 In Vivo Delivery to Epidermis 35 S...

Страница 4: ...IV Helios Gene Gun System...

Страница 5: ...elios Gene Gun is for research use only Refer to Connecting the Helios Gene Gun to a Helium Source in Section 4 for setting up a helium source and to Particle Delivery Using the Helios Gene Gun and Re...

Страница 6: ...strument 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 ge...

Страница 7: ...asier and faster to perform than the tedious task of microinjection Both transient and stable expression are possible with particle bombardment In addition to DNA RNA may also be transferred to cells...

Страница 8: ...vivo transformation Sundaram et al 1996 Keller et al 1996 Rakhmilevich et al 1996 Meristematic tissues and leaves are obvious target cells for in vivo transformation of plants Operating Principle of t...

Страница 9: ...ers from the inside wall of the cartridge As the microcarriers enter the barrel liner they pick up speed in the acceleration channel then spread out as they travel down the barrel The increased cross...

Страница 10: ...nnected while the system is still pressurized Note a check valve is also present at the female connector of the helium hose where it connects to the gene gun to shut off pressure to the gun if it is d...

Страница 11: ...microcentrifuge tubes 20 200 and 1 000 l micropipettors and tips 5 and 10 ml pipets and pipettors Lab timer Ear protection for example VWR catalog number 56610 728 earmuffs or 56610 680 ear plugs One...

Страница 12: ...ios 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 Warranty card...

Страница 13: ...ers 0 25 g 1 0 m gold microcarriers 0 25 g 0 6 m gold microcarriers 0 25 g Polyvinylpyrrolidone MW 360 000 0 5 g 5 desiccant pellets store tightly sealed 5 cartridge collection storage vials Note If a...

Страница 14: ...evice Top and bottom refer to the side of the gun that the cartridge holder is on and the side of the gun where the helium hose connects respectively see Figure 5 Fig 5 Components and controls on the...

Страница 15: ...cartridge holder from one position to the next when the cylinder advance ever is pressed Moving this bar to the left outward when inserting a cartridge holder provides additional room for maneuvering...

Страница 16: ...blade used for cutting the tubing The razor blade is held in place by the locking knob on the lock block Base The support for the arm It positions a storage vial under the tubing channels so that the...

Страница 17: ...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 Fi...

Страница 18: ...t of the gene gun as indicated by the symbol inside the battery compartment Note If the battery is inserted correctly a tone will be audible for 5 sec and the battery status LED will light if the batt...

Страница 19: ...edure 1 Secure the cylinder in a floor stand or to a wall or lab bench with a strap so it will not tip or fall during use 2 Inspect the cylinder valve for dirt dust oil grease or damaged threads Remov...

Страница 20: ...tubing installation of the nitrogen pressure regulator and connection of the Tubing Prep Station to the nitrogen regulator A peristaltic pump is recommended for removal of the ethanol from the GoldCoa...

Страница 21: ...n slide the end of the tubing support cylinder containing the O ring into the support on the motor housing Note Carry the Tubing Station by the base not by the tubing support cylinder 3 Cut a 4 5 piec...

Страница 22: ...ection 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...

Страница 23: ...n line with your fingers the pressure on the output low pressure gauge should register no more than 1 2 psi If the pressure is too high turn the regulator screw counterclockwise to reduce the pressure...

Страница 24: ...parameter optimization and starting conditions for using the Helios Gene Gun to deliver DNA to mammalian cells Procedure 1 Determining the Microcarrier Loading Quantity MLQ 1 For most systems deliveri...

Страница 25: ...at one time 50 of tubing will usually yield 80 90 cartridges Precipitation of DNA onto Microcarriers It is important to use an unopened bottle of 100 ethanol each day this step is performed Opened bo...

Страница 26: ...lumps 5 To the gold and spermidine mixture add the required volume of plasmid to achieve the desired DLR Refer to Procedure 2 for a detailed description on determining DLR Refer to Table 2 for suggest...

Страница 27: ...2 Scissors Procedure Time considerations Since only one piece of GoldCoat Tubing can be coated at a time this procedure may take 15 30 min for the first sample and 15 20 min for additional samples 1...

Страница 28: ...sier to insert into the tubing support cylinder if the end is subsequently cut in the tubing cutter Insert one end of the GoldCoat Tubing into the end of the adaptor tubing fitted to the 10 cc syringe...

Страница 29: ...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...

Страница 30: ...icrocarriers from Preparation of System Components Prior to Bombardment in Section 5 Activating the Helios Gene Gun for Gene Delivery When first setting up the system and prior to discharging the Gene...

Страница 31: ...cartridge holder is in its correct position the knob on the backside of the cartridge holder will slip into the notch on the barrel plate and the cartridge holder will be flush with the barrel plate...

Страница 32: ...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 lig...

Страница 33: ...tor by turning the regulator adjustment screw counterclockwise until the adjusting spring pressure is released and the screw moves without resistance b Release helium into the pressure regulator by sl...

Страница 34: ...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...

Страница 35: ...g Used Cartridges Depressurization and Shutdown Materials Cartridge extractor tool supplied Removing Cartridges from the Cartridge Holder 1 Hold the cartridge holder containing the cartridges to be ex...

Страница 36: ...d in Particle Delivery Using the Helios Gene Gun in Section 5 2 Immediately prior to DNA delivery aspirate the media from the dish 3 Hold the dish perpendicular to the spacer and touch the end of the...

Страница 37: ...target and discharge the gene gun Figure 18 9 Add 1 5 2 ml of media to the plate and return to the incubator In Vitro Delivery to Suspension Cultures Method 1 1 Prepare the Helios Gene Gun for operati...

Страница 38: ...pend cells in tissue culture media without serum at a concentration such that inoculating 2 ml of cells into the 35 mm tissue culture plates treated with Cell Tak will produce a monolayer 60 80 conflu...

Страница 39: ...in completely exposing the epidermis Carefully rinse the skin with warm water following depilatory treatment 4 If the target site is wet or dirty clean and dry with 70 ethanol Helios Gene Gun Operatio...

Страница 40: ...cles coated with 2 g plasmid mg gold as in Experiment 1 Prepare tubes with 0 0 05 and 0 1 mg ml PVP Bombard the target tissue using the optimum helium pressure determined in Experiment 1 Expt 3 Optimi...

Страница 41: ...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 an...

Страница 42: ...ntration on mGM CSF expression in CHO cells DNA loading rate Expression ng 106 cells 2 5 515 0 25 239 0 025 25 0 0025 0 9 CHO cells were transfected as suspension cultures using the Accell Gene Gun Ir...

Страница 43: ...25 0 5 1 2 5 0 0 1 300 500 Pig7 8 10 Inner thigh clipping not necessary 0 25 0 5 0 5 2 5 0 05 500 Rabbit27 Back 0 5 1 2 None 350 400 Do not use a depilatory agent to remove the fur Section 6 3 step 3...

Страница 44: ...e in place and are not worn Check that the barrel liner is tight see Replacing the O Rings and Barrel Liner on the Helios Gene Gun Section 10 Check that the cartridge holder is properly positioned in...

Страница 45: ...alog 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 1652417 Barrel Liner 5 1652426 Cartridge...

Страница 46: ...ery 1652423 Razor Blades 10 1652416 O rings gene gun 1652417 Barrel Liner 1652426 Cartridge Holder Gene Gun Specifications Specifications Physical Dimensions Approximately 8 x 10 Weight 3 15 lb Constr...

Страница 47: ...tro using the Helios Gene Gun System Qiu et al 1996 The procedure is similar to that for DNA but the precipitation step is performed with ammonium acetate and 2 propanol Materials In addition to those...

Страница 48: ...cartridge holder from the gene gun 2 Place the barrel liner into the gene gun and turn clockwise until the threads just bind Do not overtighten The O rings function to keep the helium from expanding a...

Страница 49: ...Figure 23 1 Remove the tubing support cylinder by pushing it to the right to contract the spring on the support bar then raise the left side up and away from the base of the unit 2 The outer O ring sh...

Страница 50: ...ubing cutter arm to the right with the index finger of your left hand This will disengage the arm from the pivot pin on the left side of the base and from the retaining pin on the right side of the ba...

Страница 51: ...g cutter may be cleaned using a cloth and a mild soap solution Do not autoclave the tubing cutter Although GoldCoat Tubing may be autoclaved this is not recommended since it may result in water conden...

Страница 52: ...tridges should be completely empty Spot darkness Proportional to particle penetration Spot width Proportional to particle spread Spot shape Uniformity of shot Discharge into 3 Water Agar Materials Sli...

Страница 53: ...ion pattern of the microcarriers is bell curve shaped with respect to depth and area The highest microcarrier density is concentrated at the center of the shot and at the surface of the target site Fo...

Страница 54: ...es Microfuge Procedure 1 Place five 0 5 cartridges in a microcentrifuge tube and add 500 l of TE 2 Sonicate and or vortex to dislodge gold and solubilize the DNA Spin for 5 min in a microfuge to pelle...

Страница 55: ...Ther 7 1535 1543 McCabe DE et al 1988 Stable transformation of soybean glycine max by particle acceleration Bio Technology 6 923 926 Qiu P et al 1996 Gene gun delivery of mRNA in situ results in effic...

Страница 56: ...shots by engaging the safety interlock and firing the trigger Firing the Device 1 Load cartridges into the cartridge holder and place in gene gun 2 Prepare position target cells for bombardment 3 Bomb...

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Страница 60: ...61 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 310 318 540 666 New Zealand 64 9 415 2280 Norway 47 0 233 841 30 Poland 36 01 459 6191 Portugal 351 21 4727717 Russia 7 495 721 14 0...

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