Gene gun technology for delivering genes into the skin
Gene gun technology for delivering genes into the skin
Gene gun technology is a fast and simple technique for delivering genes into cells. This technology has many advantages: it allows direct delivery of plasmids without the need to construct complex biological vectors as is the case with some viral vector systems. In theory, DNA can be delivered to any cell using the gene gun technique. DNA can penetrate cell walls, cell membranes, stratum corneum, or other protective structures, and delivery is not limited by cell surface receptors or molecules.
Author: T. Friedman et al., Translated by W. Jing et al. This experiment is from "Gene Transfer".
Operation method
Delivering Genes with H e l i o s Gene Guns Move Delivering Genes with H e l i o s Gene Guns material reagents Experimental animals (usually mice weighing about 20 g) CaC l2 (I.0mol/L) 0,22um filter for sterilization, store at room temperature. Ethanol (100% anhydrous ethanol; Sp e c t r u m ) To avoid water absorption, be sure to screw the cap on tightly when not in use. Plasmids expressing the target gene Empty vector plasmids were used as a blank control, if required. Plasmids are amplified by standard methods (SatnbrookandRussell2001) and purified using commercial kits (e.g., Q uantum Prep or A urutn columns, Bó Lè products). Resuspension in T E liquid Arginine (50m m ol/L , free base; Sigm a-A ld rich ) Dissolve I g of linamine in 6.8 ml of sterile Milli-Q water, filtered by 0.22um membrane to make lmol/L storage solution. The solution was filtered with 0.22um membrane to remove bacteria, and then made into lmol/L storage solution, which was stored at 70℃. Dilute the stock solution with Milli-Q water at 1:20 to 50 mmol/L working solution and store at -7 CTC. Instrumentation Analyzing Balance Electric shaver Helios Gene Gun System (Bio-R a d Laboratories) The system consists of the HeIios Gene Gun, sample tube holder, gasket and O-ring, 9-volt battery, sample tube extraction tool, special helium valve and regulator, sample tube preparation station, nitrogen valve, IOml syringe with silicone rubber adapter tube, gold-plated tubing (Tefzel) tubes, 1.0um and 1 . 6 um gold pellets, sample tube cutter, polyvinylpyrrolidone, desiccant, sample tube reservoir. Compressed helium (4. 5 grade) Compressed nitrogen (4.8 grade) with 1 to 3 psi regulator. Constant Flow Pump (optional) Im l syringe with 25-gauge half-inch long needle Ultrasonic water bath method Preparation of DNA-coated gold particles The following describes a method for preparing DNA-coated gold particles that are large enough to fill a long sample tube, which can typically be cut into 40 to 45 half-inch long sample tubes (Steps 21 to 23). 1- Add 25m g of gold pellets to a 1.5m l centrifuge tube. If a control sample tube is required, weigh an additional 25m g of sample and place in another 1.5m l centrifuge tube. The two samples are processed in parallel except for the addition of the plasmid. In order to check the background level of the target gene in the experiment, gold pellets without DNA or gold pellets coated with an empty vector that does not express the target gene can be prepared as a control. Repeat steps 12 to 16 to load these gold particles into another Tefzel. 2 . Add IOOfxm 50m m o l / L of spermidine solution to each centrifuge tube. 3 . The mixture of gold pellets and spermidine was vortexed for a few seconds, and then sonicated in a water bath for 3~5s to obtain well-dispersed gold pellets. 4 . Add 50um 1 ug per micrometer of plasmid to the above gold particles solution to give a D N A loading ratio of 2, while the control vector is properly prepared. 5 . Vortex the DNA and gold particles at high speed for about 5 s. 6- While vortexing at a moderate rate, carefully open the cap of the centrifuge tube and add a slow drop of IOOuI l m o l/L CaCl2 solution to the mixture. Cap the tube and vortex at high speed for 5s. 7- Allow the gold pellet carrier to stand for l O m i n at room temperature. 8 . Centrifuge the gold pellet carrier for 15s and discard the supernatant. 9- Slightly vortex the centrifuge tube so that the precipitate is resuspended in the remaining solution. 10- Add I m l 1 0 0 % ethanol to the centrifuge tube. Centrifuge for 5s, discard the supernatant and repeat twice. 1 1 . Slightly scour the tube so that the precipitate is resuspended in the remaining solution. Transfer to a 15 ml capped polypropylene centrifuge tube. Wash the 1.5 ml centrifuge tube twice with 200 ul of 100% ethanol and combine into a 15 ml centrifuge tube. 12. Add 2.6 ml of 100% ethanol to the 15 ml centrifuge tube and immediately prepare the sample tube with this suspension. Transfer the DNA gold particle carrier suspension to the sample tube. 13 - Blow the TefzeI tubes with nitrogen to completely dry them. Insert the unsheared TefzeI tube through the tube support ring from the left distal O-ring to the right of the sample tube preparation station. Turn on the compressed nitrogen and adjust the pressure to 1-3 psi, then adjust the flow rate to 0-5L/million using the adjustment knob on the flow meter and vent for 15 minutes. 14 . Attach the I O m l syringe with the 1 8 ft silicone rubber adapter tube to the syringe sleeve and secure it to the syringe holder at the bottom of the Sample Tube Preparation Station. Remove the Tefzel tubing from the Sample Tube Preparation Station. Turn off the nitrogen using the adjustment knob on the flow meter. If removing ethanol from the Tefzel tubing with the worm pump, first set the worm pump speed to 3.6 to 7.2 ml/min with ethanol. connect the pump to the Tefzel tubing with a silicone rubber adapter tube. The slower the rate of ethanol removal, the more ethanol will be removed from the Tefzel tube. 15- Cut a 30in (75c m ) length of Tefzel tubing with a cutter (the control needs to be the same length). Ensure that neither end of the 30in long tube is twisted out of shape. Attach one end of the 30-in long sample tube to the silicone rubber adapter tube with the IOM syringe attached. Push the syringe cartridge all the way down before connecting. 16. Vortex the gold pellet carrier suspension (if desired, sonicate to obtain a homogeneous suspension). Resuspend the gold pellet by inverting the centrifuge tube containing the gold pellet carrier several times, uncapping the tube, inserting the free end of the sample tube, and then using the syringe at the other end to rapidly aspirate the suspension of gold pellet carrier into the sample tube (approximately 2in, 58c m deep). Do not vortex the Gold Pellet Carrier Suspension as it is drawn into the sample tube. Do not attempt to aspirate all of the ethanol from the centrifuge tube. Special note: Do not inhale air bubbles. I7- Remove the sample tube from the centrifuge tube, flatten it, and continue to aspirate the suspension into the sample tube for 2 to 3 in. Immediately load the sample tube containing the gold pellet, along with the syringe, into the correct position on the sample tube preparation station. 18- Allow to stand for 3~5m i n . Disconnect the sample tube from the silicone rubber connecting tube and attach it to the silicone rubber connecting tube of the I O m l syringe that has been secured to the bottom of the sample tube preparation station. Use the syringe to draw off the ethanol at a rate of 〇.5 ~lin/s (it takes roughly 30 to 60s). If using a peristaltic chestnut, attach the pump with the adjusted flow rate to the sample tube. This makes it unnecessary to place an IOmI syringe on the syringe holder. 19- Disconnect the syringe and silicone rubber connection tube from the sample tube. Immediately rotate the sample tube in the Sample Tube Preparation Station by 18 °. 3 to 4s later turn on the Sample Tube Preparation Station power to rotate the sample tube. 20. After rotating the sample tubes for 20 to 30 seconds, open the control valve on the nitrogen flow meter and blow-dry the sample tubes with nitrogen at a flow rate of 0-35 to 0.4L/m i n while the tubes are rotating. Continue this process for 3 to 5 m i n before turning off the sample tube preparation station motor switch. Close the nitrogen flow control valve and remove the sample tube. Preparing Sample Tubes for the Helios Gene Gun 21-Inspection of the sample tube to determine if the gold particle carrier is evenly distributed. Use a marker to mark the tube where the gold particles are not evenly distributed. Uneven distribution is usually confined to the outer 2in of the gold particle sink, but can sometimes be found inside the sample tube. Ideally, the particles should be uniformly distributed over the surface of the inside of the sample tube; however, it is possible to blow the particles to one end of the tube during the nitrogen blow-drying process. However, as long as the naked eye can determine that each 0.5in long sample tube contains the same amount of gold particles, it can be used to prepare the sample tubes for the Gene Gun. 22. Use scissors to cut off any uneven sections. Use a sample tube cutter to cut the remaining portion into small sections of 0.5i n . 2 3 . Store the sample tubes in a sample tube storage jar with desiccant. Cap the jar tightly, mark it, seal it with a sealing film and store it at 4°C . The tubes can be stored for up to one year under the above conditions. Transfer of pellets with H e l i o s Gene Guns This gene gun can be used to transport DNA to a variety of cells in vivo; however, it is easiest to transport DNA to epidermal cells. By adjusting the helium pressure, it is possible to deliver most of the particles to the epidermal cells under the stratum corneum without penetrating the dermal tissue. 2 4 . To activate the Helios gene gun for gene transfer, load the gene gun with a battery and metal washer. Attach one end of the helium hose to the Helios Gene Gun and the other end to the helium control valve. Open the main valve of the helium cylinder and adjust the helium regulator to the proper pressure. For example, for gene transfer into mouse skin, a pressure of 350 psi is usually appropriate when using 1.0 to 1.6um gold particles. 2 5 . Attach an empty sample tube rack to the gene gun and make one or two shots to pressurize the system. Remove the empty sample tube rack from the gene gun. 26. Load the sample tubes prepared in steps 21 to 23 into the sample tube rack. Insert this sample tube rack into the Gene Gun. Animal gene transfer 27- Select the appropriate anesthetic to inject into an animal to anesthetize it based on the I A C U C operation. It is recommended to inject 200ul of a mixture of mebendazim (lmg/ml) and ketamine (lOmg/ml) into the peritoneal cavity of a 20 g weight mouse. 28. Shave the animal's target area, usually the chest and abdomen, and brush it. The backs of the ears are also a good site and do not need to be shaved. 29. Place the gene gun on the target site and fire. 30. After an appropriate period of time, the transhuman gene is analyzed for expression using appropriate analytical methods. For more product details, please visit Aladdin Scientific website.
Narcotics ([xylazine (lm g/m l) and/or ketamine (10m g/m l)].
Plasmids were resuspended to 0.5-2 mg/ml.