Among the various viral and non-viral means commonly used for nucleic acid transport and gene expression in animal cells, the naked plasmid D N A technique has many advantages. First, plasmid D N A can be operated directly by D N A recombinant technology without special requirements for technology and equipment! Second, this method is not costly even for large-scale production; third, because plasmid D N A is not immunogenic, it can be administered multiple times without inducing antibody production (Jia〇et al. 1992). Contrary to common perception, even if the exogenous gene is not integrated into the chromosome, it may still be possible to obtain long-term expression in dividing or slow-fe dividing cells by means of the naked plasmid D N A technique. Although the transfer of nucleic acids by nonviral routes has long been considered inefficient, the level of naked D N A expression by intravascular transfer has approached that achieved with viruses as vectors. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Plasmid DNA was injected into the liver of mice by localized pressure injection into the tail vein. Move Plasmid DNA was injected into the liver of mice by localized pressure injection into the tail vein. Material reagents Preparation of IOX storage solution with deionized water: 0.85 % NaCl, 0.5 % NaCl, 0.5 % NaCl and 0.5 % NaCl 85 % NACL, 0.03 % KC1 < ! 0.03 % KC1 < ! > 0.03% KC1 < ! 0.03 % CaCl2 < ! > Filter Mouse IC R or C57BL/6 mice are the main experimental animals. Other strains such as BABL/c, ddY and transgenic mice are also available. Plasmid D N A Plasmid DNA should be endotoxinized for best results. Endotoxin-removed plasmid DNA is extracted using kits available from QIAGEN et al. Endotoxin-containing plasmid samples should be processed using purchased endotoxin-removal kits. Avoid using T ris-containing buffers in the final step of plasmid extraction because of their toxicity. Transcriptional regulators such as enhancers and promoters on the plasmid should be selected correctly, as they can affect the number and duration of transgene expression. For example, plasmids containing viral promoters (e.g. cytomegalovirus early enhancer/promoter) can be highly expressed in the early stages of liver transfection, but the expression can be terminated within 24 hours. Plasmids that contain a long-term expression element in liver cells have been reported (Miaoet al.2000; Woidelletal.2005). Instrumentation Conical tube (plastic, 50 ml; see step 1) Disposable syringe (3 ml) Heating lamp Injection Needles (#2 7) Methods 1. Mice were placed head-first in 50 ml conical tubes with 3-5 mm breathing holes in the bottom and a slit in the cap to allow the tail to pass through. Specialized fixation tubes for mice or rats can also be purchased. 2- Before injection, irradiate the mice with a heat lamp for IOmin to dilate the blood vessels in the tail. Pay attention to the position of the heat lamp, do not overheat the mice too close. Because the tail vein is visible, the desired injection effect is ensured. In black mice, the tail vein is more difficult to see. 70% isopropyl alcohol can be applied to the tail to increase the contrast between the blood vessels and the skin. 3. Add plasmid DNA (usually 5-100Mg, endotoxin removed) to a sterile IXRignger solution at room temperature. The total amount of solution injected is approximately 10% of the body weight of the mouse (e.g., 2 ml for a 20 g mouse). Insufficient injection volume will result in poor plasmid transfer. Salt solution should not be used as the injection solution because it can cause post-injection syndrome. The amount of nucleic acid in it can be proportionally increased or decreased when the injection volume is certain. 4- Attach a 0.5-inch long 27-gauge needle to a 3-mil syringe and insert the needle into the dilated caudal vein, preferably in the middle of the tail or near the distal end. It is best to insert the needle all the way into the vessel to avoid leakage. 5. Push a small amount of injection fluid slowly to ensure that the needle has penetrated the vessel. After making sure that the needle is inserted correctly, all the fluid should be injected into the tail vein within 5~7s. The ability to push the injected fluid as quickly and evenly as possible into the animal's vasculature has a significant impact on the ability to maximize the efficiency of the infusion. After injection, the animal may appear to be stationary and dyspneic for a short period of time, but usually not more than 15~20 min. reagents Isoflurane rat Hot water (see step 4) Apparatus Butterfly Infuser (21 gauge X 3/4i n syringe needle) Disposable syringe (60m l ) Dissecting microscope Harvard Apparatus P H D 2000 Syringe Pumps Surgvet/Anesco Isotec 4 Flow Meter METHODOLOGY 1. Dissolve plasmid DNA (50-500 ug, endotoxin removed) in 10% of the body weight of the rat in a sterile, room-temperature IXRigner solution (e.g., 120 g of rat injected with 12 ml of solution). 2.60 ml syringe to draw up the injection solution and connect it to a butterfly infusion set (2 No. 1 winged infusion needle). 3. Set up a Surgvet/Anesco Isotec 4 flow meter and anesthetize the animal with 1 % to 2 % isoflurane (○.4L/m i n ) . 4- The rat's tail was immersed in hot water to vasodilate the blood vessels. 5. Under a dissecting microscope, bevel the tip of the 21-gauge winged infusion needle at the end of the infusion set to enter the needle. Place the syringe in the setup Surgvet/Anesco Isotec 4 flow meter. 6. Inject a small amount of fluid first to ensure the needle has penetrated the blood vessel. After ensuring that the needle is feeding correctly, inject the injection solution at the syringe pump's maximum injection rate (l O O m l/m i n ). If the syringe pump is not available, manual injection may also be used, but its effectiveness may be impaired. reagents Isoflurane saline (medicine) 0 . 9 % NaCl, prepared with deionized water. Filter to remove bacteria and store at room temperature. Ketoprofen or acetaminophen (see step 9) mice I C R or C 57B L/6 mice are primarily used as experimental animals. Other strains such as B A B L /c, d d Y and transgenic mice are also available. Plasmid D N A Same as the preparation method for Plasmid D N A used for tail vein hyperbaric injection (Scheme 1). Unlike liver, plasmids for long-term expression in muscle can contain viral or recombinant exogenous enhancers/promoters. Maximum intramuscular expression is not achieved until 4-7d after injection. Instrumentation Disposable syringe (I O m l) Dissecting microscope material (optional) Tweezers Gel Foamer H a rvard Apparatus P H D 2000 Syringe Pumps Medical Tape Needle Holders lancet small traction device Surgvet/Anesco Isotec 4 Flow Meter Surgical Lines Syringe (#30) Reduce the outer diameter of the needle by sharpening it with a Drummel tool. Tourniquet, hemostat To make a rat or mouse tourniquet, simply and efficiently cut off the tip of one finger of a latex glove and then cut 1 cm off the end (see Step 2). METHODS 1. Set up a Surgvet/Anesco Isotec 4 flow meter and anesthetize the animal with 1 % - 2 % isoflurane (0.4 L/min) to the desired depth for surgery. 2- The lower limbs of the animal to be injected are debrided and tied with a tourniquet to stop the flow of blood in and out. Thread the leg through the tourniquet until the proximal end is tightly wrapped (only the proximal end of the quadriceps muscle of the leg should be partially applied). Tighten the tourniquet and secure with hemostatic forceps . The animal's limbs are secured to the surgical table with medical tape, keeping the animal in a supine position. There is a relationship between the area of nucleic acid delivery or arrival and the position of the tourniquet. 3 . A small midline incision is made in the center of the leg near the ankle to expose the saphenous vein. 4- Dissolve the plasmid D N A (usually 5 to 300ug g , de-endotoxinized) with 1.0 m l room temperature, sterile saline. This injection dose is optimal for 2 5 g animals, but the different body weights of the animals have little effect on the dose. 5- Draw up the injection with a 3 m l syringe and attach a P E -I O catheter. A needle (30-gauge) is fitted to the catheter and inserted into the saphenous vein using a needle holder. A dissecting microscope makes needle insertion easier. The needle can be kept in place by applying light pressure with a cotton swab. Care must be taken to keep the leg moving during the injection, otherwise the blood vessel may be easily punctured. 6. Set the Harvard Apparatus P HD 2000 syringe pump so that the injection is completed at a rate of 8 ml/m i n . Leave the tourniquet in place until 2m i n after the injection, after which the catheter and tourniquet are removed. Hemostasis was achieved by gel foam and light pressure. 7- Close the wound with #4-0 suture. 8 . Observe the animal closely until it is awake from anesthesia, and use a heat lamp to prevent loss of body heat to facilitate recovery. 9. Ketoprofen (5mg/kg) is injected subcutaneously daily for 2d postoperatively. Acetaminophen [lOOmg/(kg.d), taken orally with water] can also be used instead of ketoprofen. Acetaminophen [lOOmg/(kg.d), orally with water] can also be used instead of ketoprofen. Instrumentation Butterfly Infuser (25 gauge X 3/4in needle) Disposable syringe (IOml) Method 2. An IO ml syringe is drawn up and attached to a butterfly infusion set (25-gauge winged infusion needle). 3 . Insert the needle obliquely below the lateral saphenous vein. 4 - Set the Harvard Apparatus P H D 2 0 0 0 syringe pump so that the injection is completed at a rate of lOnu/m i n . For more product details, please visit Aladdin Scientific website.
Ringer's solution
Dilute with sterile deionized water as working solution and store at room temperature.
If the body weight of the rat is more than 200 g, the injection volume should be reduced appropriately, and the total amount should not exceed 18 ml.
It makes the veins easily visible and facilitates needle entry.
1 . Dissolve plasmid D N A (50 ~ 750 ug, endotoxin removed) in ○. 0.3 times the body weight of the rat in sterile, room-temperature saline (e.g., 120 g rats injected with 3. 6 m l of fluid).