Experimental procedure 1. Murine cloning 1) Detection of estrus period: In the establishment of transgenic animal models, the monitoring of estrus period of female rats has high skill. The estrus period of mice is divided into four periods: a. Late estrus: no expansion of the vaginal opening, the surrounding tissue is gray, no swelling around the vaginal opening b. Intercourse: The vaginal opening is small, and the surrounding tissue is gray or blue. c. Early estrus: the vaginal opening gradually expands, and the tissue around the vagina changes from pink to red. d. Estrous period: the color of the tissues around the vagina changes from red to pink, streaks are visible on the dorsal lip of the vaginal opening, swelling is visible on the ventral lip of the vaginal opening, and there is extravasation of secretory fluid. Randomly select animals. At any time, 20% -25% of animals may be in estrus. The estrous period of most rodents lasts 4-5 days on average, so after synchronizing the estrous cycle of individuals of social animals, a large number of estrous animals may be produced in any period. The selection of animals during estrus requires two indicators: the color of the tissue around the vagina and the degree of tissue expansion. The vaginal tissue of animals during estrus is dark pink, but it is not the same as the color of inflammation. In addition, the dorsal and abdomen tissues around the vaginal opening were swollen and shiny, with stripes initially visible on the abdomen. Individual vaginal tissues are darker in color, but there is no swelling, or the vaginal opening is slightly enlarged and slightly gray is non-estrous animals, do not mistakenly choose them for mating. 2) Confirmation of mating: Yin plug formation. After the animals are caged, it is necessary to determine whether the male individual successfully mates with the female mouse. Because male semen can solidify into a soft suppository in the vagina, a vaginal suppository is formed shortly after successful mating. Use the middle finger, little finger and thumb of the hand to pinch the base of the female tail so that the head of the mouse is down. Carefully observe the vaginal opening. The mated female mouse can see a white eraser-shaped vaginal plug, which is easy to see with the naked eye When confirming, a small probe can be used to check whether there is an embolus in the deep vagina. Checking the suppository should be done early in the morning every morning, because over time, the suppository will be expelled from the body. 2. Vasectomy The vas deferens mice are used to mate with the embryo transfer embryos to make them pseudo-pregnant. Mice undergoing vas deferens at 6-8 weeks of age, CD-1 B6D2F1 (C57BL6 * DBA / 2) or Swiss Webster are the usual subjects. The anesthetic dose of avertin for intraperitoneal injection is 240 mg / kg. 1) After weighing, anesthetize the mouse, fix the mouse in the dorsal position, cut the hair with large scissors close to the skin, apply 70% ethanol to sterilize the surgical incision to prevent the hair from contaminating the incision. 2) Cut the skin of the lower abdomen approximately 1.3cm in front of the genitals, make an incision of about 1cm, and wipe the incision with 70% ethanol-soaked gauze to clean the hair. 3) Cut the peritoneum and position the bladder. There is a tube running on each side. Use tweezers to gently hold the left tube. Lift the part to make the surgical field clearly visible and confirm it as a vas deferens. 4) Insert the forceps under the vas deferens to keep them in a natural state with their ends vertical. At the same time, the vas deferens are ligated with sutures at both ends at a distance of about 4-5 mm. Cut the vas deferens between the two ligation points and place it on the gauze to confirm the operation on this side. 5) Gently put the two broken ends of the vas deferens back into the abdominal cavity and treat the right vas deferens as above. After the surgery on both sides, the abdominal wall incision was sutured with 2-3 separate sutures. The sutures to be used must be soaked in 70% ethanol to keep the sutures moist and prevent tissue adhesion during ligation. Close the skin with two or three automatic clips. 6) To keep the mouse wrapped in gauze or place it in a thermal pad to keep it warm, the mouse under anesthesia should be closely monitored until it is fully awake. After the operation, the mice were fed for 2 weeks to determine whether the operation was successful. 7) Experimental feeding: 1-2 female mice were bred with the vas deferens in a cage, and the vaginal suppository was examined the next morning. Female rats with vaginal suppositories flushed their fallopian tubes 24 hours after treatment with phosphate buffer. The egg cell should be in the single-cell stage or infertile state. If it is in the two-cell stage, the vas deferens are not completely removed, and the male mice should be rescreened. 3. Collection of fertilized eggs 1) Collection of fertilized eggs: There are three methods for obtaining fertilized eggs: natural ovulation, hormone-induced ovulation, and in vitro fertilization. Induced ovulation method: Female mice reach sexual maturity 6-8 weeks after birth, and the sexual cycle is 4-5 days. The ovulation time can be adjusted by the brightness and darkness of the breeding room, so the light and dark rules of the breeding room must be accurate and strict地 管ç†ã€‚ Local management. The sexual cycle is the result of the interaction between follicle stimulating hormone and luteinizing hormone, and we can give these hormones from outside to induce ovulation. After intraperitoneal injection of 5 international units (IU) of pregnant horse serum gonadotropin (PMSG) into mature female mice, approximately 48-54 hours later, 2.5-5.0 IU of human chorionic gonadotropin (hCG) was injected into In the abdominal cavity of the same mouse, ovulation can be induced after about 12 hours. The number of ovulation can be up to 2 times of natural ovulation, the effect is very good. Female mice should be caged with males immediately after giving hCG. Mature male mice should be housed in one cage per cage. Male mice range in size from 12 weeks to 1 year. When closing the cage, the hormonalized female mice must be placed in the cages of male mice. Male mice release estrogens that promote female estrus. It is recommended not to change cages during the mating process. Male mice that have been mated will be mated for the next time after a week. Micro-injected fertilized eggs are best collected a few hours before the next morning after mating. At this time, the injection operation is easy. The vaginal plugs of mice are easy to see after mating overnight and can be indicated by mating indicators. It is generally normal to count vaginal suppositories in superovulated mating females. A low rate of thrombogenic female mice indicates that the gonadotropin has lost its effectiveness or that male mice have overmated or aging male mice. Harvesting fertilized eggs must open the abdominal cavity of the mouse, and the fallopian tubes must be carefully cut open. Tubal irrigation or tubal incision can be used as a method of collecting fertilized eggs. 2) Incision of the fallopian tube in the abdominal cavity a. Quickly kill the mice by breaking the neck or inhaling carbon dioxide. b. Fix the animal's dorsal position on sterile dry absorbent paper, cut the hair and thoroughly apply the surgical site with 70% ethanol. To avoid hair contamination of the surgical field. c. Hold the midline skin of the lower abdomen with ophthalmic forceps, make a small transverse incision with surgical scissors, bluntly separate the scissors to fully expose the surgical field, or bluntly tear the skin to expose the peritoneum. Open the peritoneum with ophthalmic scissors to fully expose the surgical field of view of the abdominal cavity and uterine horn. The uterus is Y-shaped, a muscular organ that starts from the pelvic bladder, branches upward to the uterine horns on both sides, and traverses upward into the abdominal cavity. d. Use forceps to hold the uterine horn 6-7mm away from the fallopian tube ovary, flip it gently, and then pull it out of the abdominal cavity. Break the junction of the uterine horn and the fallopian tube. e. Use forceps to pull out the fat pad, ovaries, fallopian tubes and uterine components. Carefully cut off the thin film between the ovary and fallopian tube, then cut off the fallopian tube and part of the uterine horn, place it in a small glass dish containing working fluid, and repeat the above operation on the other side of the fallopian tube. An animal. 3) Collection of fertilized eggs in isolated fallopian tubes: Observed under the dissecting mirror, there is obvious flushing near the upper part of the funnel of the fallopian tube. This is the ampulla. Using ophthalmic forceps to tear open the enlarged ampulla, you can see the cluster of mound cells surrounding the fertilized egg. a. Transfer the fallopian tube to a small glass dish containing working fluid. b. Ophthalmic forceps hold the ampulla, and use another ophthalmic forceps to tear open the enlarged fallopian tube. The free fertilized egg slowly flows out. You can also use forceps to gently squeeze the fallopian tube to push the fertilized egg out of the crack. 4) Hyaluronidase treatment and rinsing of fertilized eggs The fertilized egg in the working fluid may be clumpy. The fertilized egg for microinjection must be a single cell without cell debris. When rinsing the cells, first remove the cell debris with the working solution. Non-clumped cells can be collected in a glass dish containing working solution with a sterile pipette, pay attention to the tension of the pipette. The hyaluronidase dissolved in the working fluid must be closely observed when digesting the cell mass and the complex. Once the cell mass is dissolved, the single cells are transferred to the fresh working fluid immediately to prevent excessive digestion. Rinse with working solution 2-3 times. After removing the debris, place the fertilized egg in a special medium (M16) with a sterile pipette, put it in a 37 ° C, 5% CO2 incubator to be injected, the medium is covered with an upper layer High-pressure sterilization of mineral oil can prevent pollution and prevent the evaporation of medium water to affect pH. Fertilized eggs develop faster in vitro than in vivo. During the incubation process, pay attention to observe that some fertilized eggs clearly show the formation of pronuclei. During this period, eggs with clear pronucleus (a little irregular shape) can be selected for injection, and other eggs continue to be cultured. After injection, re-screening and re-injecting until a satisfactory number of injected eggs are obtained. 4. Microinjection 1) Preparation of introduced DNA: Microinjection first involves the preparation of introduced DNA. The transferred gene for microinjection is usually linear DNA with the vector sequence removed. The vector used for transgene is a eukaryotic expression vector, that is, contained in mammalian cells Expressed eukaryotic promoter. The so-called tissue-specific realization is mostly through tissue-specific promoters to achieve tissue-specific expression. To prepare transgenic mice, the DNA to be introduced must be separated and purified. The experiment must use DNA that has been identified by agarose electrophoresis to determine its purity. There is no specific limit to the size of the introduced gene, and long-chain DNA can also be successful. In the experiment, pay attention to prevent some impurities from clogging the injection needle, such as agarose particles, fiber materials, etc., as far as possible to remove ultracentrifugation. The quality of DNA injection is the key to the success of the experiment. Studies have shown that the initial concentration of DNA injection is about 1 ng / ml. When the DNA injection concentration exceeds a certain upper limit, the experimental effect is not good, and more than 5 ng / ml will produce significant toxicity. The preparation procedure of the introduced DNA is as follows: a. Separate the DNA to be inserted from the carrier by agarose gel electrophoresis in Tris / acetate / EDTA buffer and stain with 5mg / ml ethidium bromide. b. In order to prevent the destruction of ethidium bromide inserted into the DNA, develop with long-wave ultraviolet light. c. Cut the gel slice where the gene of interest is located, and prepare the target DNA by electrophoresis, or use Qiaex gel extraction kit for extraction. d. Ethanol precipitates the target DNA. Add 1/10 volume of 3M sodium acetate to the sample, mix well, then add 2-2.5 times volume of sterile 100% ethanol for precipitation. e. After incubating at -20 ° C overnight, centrifuge at 10,000 rpm for 5 minutes, collect the pellet, and resuspend the pellet in Elutip buffer. f. Treat the target DNA with Elutip-D mini-column. g. Re-precipitate DNA according to step 4, rinse the precipitate with 70% ethanol 2-3 times, and dry the precipitate in vacuo. The cleaning and drying process is extremely important because residual salt and ethanol are fatal to the development of fertilized eggs. h. Injection buffer (10 mmol / L Tris-HCl / 0.1 mmol / L EDTA, pH 7.5) to resuspend the pellet, the buffer must be prepared with Milli-Q purified water i. Evaluate the concentration of the target DNA by fluorescence photometer or gel electrophoresis colorimetry j. Adjust the concentration of the target DNA to 5-10ng /? l with injection buffer. 2) Instrument preparation a. Making of injection needle The injection needle is a thin-walled capillary pipette containing glass filaments, which can be loaded by capillary siphoning. Use a needle puller (SUTTER, P2000 laser based micropipette puller, see the product manual for specific operating procedures) to pull the injection needle horizontally or vertically. It must be ensured that the prepared capillary can enter the needle puller so that the heating assembly is approximately in the center of the capillary. Generally, a micro-electrode tube with an inner diameter of 1.0 mm is used as a material for the injection needle. The micro-electrode tube is available for purchase, and it is compatible with a needle pulling instrument. In addition, the setting of the needle puller should be selected so that the filament temperature and pulling force (primary and secondary tension) will be in the best state. Specific needs pre-experiment to determine its optimal setting. The injection needle to be used should be strictly washed with distilled water to remove residual carbonized particles. Strict experiments Microelectrode tubes should go through the procedures of sour acid, soaked distilled water and siliconization before use, but some people omit this step and have good results.The needle cannot be cleaned after the needle is pulled. No carbonized particles will remain. If the needle tip is cleaned after pulling, it will be easily broken. Narishige Japan model PN-30 can be used. b. Making oviduct In order to avoid mechanical damage, the mouth of the oviduct should be a blunt end and its pores are limited, so that the fertilized egg gently attaches to the negative pressure pipeline. This high density can resist the damage of the sealing system, and the seal can reduce the rotational movement of the fertilized egg injection. The mouth of the oviduct should be smooth, flat and perpendicular to its long axis. Specific preparation operation: Hold both ends of the capillary glass tube with both hands, place the alcohol flame in the middle of the tube and burn red until it becomes soft, and then leave the flame. At the same time, stretch out the hands with both hands and thin the soft part. Carefully cut the thin tube with a glass knife or fine sand wheel, and the incision should be flush under the microscope (if not, it should be cut again). Then passivate the incision at the edge of the blue flame at the bottom of the flame of the alcohol lamp (that is, burn the nozzle at the edge of the blue flame briefly, and then check the shape of the nozzle under the microscope, and repeat it until you are satisfied). If the laboratory is equipped with an oviduct holder, you can directly monitor the shape of the hot filament burning the oviduct tube mouth under the microscope, adjust the relative position between the two in time, and it is easier to get a satisfactory oviduct tube. The oviduct should be adjusted, and the end of the oviduct should be bent to 15 degrees (different microinjector degrees may be different, bent to about 25 degrees, generally 20-25 degrees) slightly bent to facilitate use. The oviduct is made of glass without filament, and the outer diameter of the oviduct should be 100-140um under the microscope with a graduated eyepiece. The inner diameter of the oviduct is very important. The platinum filament can be used to burn the orifice so that the inner diameter is about 30-50um. The inner diameter should be able to suck the fertilized egg without sucking the egg into the tube. For ease of operation, the oviduct can be further adjusted so that its tip is slightly curved (15 degrees). c. Production of oviduct Light the alcohol lamp and adjust the flame to the best. Hold the glass capillary pipette or pasteur pipette and rotate the flame over the flame. When the straw begins to soften, quickly evacuate the flame and pull it outwards to make the straw longer, forming a straw with a diameter of about 200ul. Pay attention to the time of leaving the flame and the pulling force during the preparation process, and try to ensure the consistency of the preparation straw. To score the straw, gently break the straw to see if it sounds crisp, or pull the straw or just bend it until two straws with the same score break. Check the suction tube under the dissecting mirror (to be on the fuse) and adjust it to make sure its caliber and mouth are smooth and flat. d. Production of oviduct The process of making a straw for transferring fertilized eggs into pseudo-pregnant mice after injection is the same as washing the egg tubes. The difference is that the diameter of these straws is slightly smaller, about 150um (150-160um), which is slightly larger than the diameter of a single fertilized egg, which will cause the egg cells to fill the transfer tube finely and transfer to the fallopian tube. During the polishing process, the tube mouth is required to be smoother and smoother to reduce the damage to the fallopian tube when it is inserted into the fallopian tube. At the same time, it must be noted that the tube tip should not be too long on the flame to prevent melting and blockage. The nozzle should be flat and passivated. e. Preparation of concave slides A concave slide suitable for carrying fertilized eggs for microinjection must be prepared as a microinjection tank, and a petri dish can also be used as an injection tank. The fertilized egg on the slide should be infiltrated in a pH buffered working solution, such as M2 solution, to make the fertilized egg Keeping 30-40 minutes outside the incubator can be protected. The specific concave glass preparation procedures are as follows: Use a manual pipette to add M2 solution to the base of the concave glass slide hole in the ultra-clean table to form a liquid surface with a diameter of about 0.6cm. The liquid surface of the droplets should be horizontal and flat to avoid the refraction effect of the liquid. Pipette the mineral oil used for embryo experiment on the M2 solution. The amount of mineral oil should just reach the highest level of the M2 solution. Place the concave glass slide on the stage of the inverted microscope and adjust the focal length under the low magnification lens to make the M2 The bottom surface of the solution droplet is clear. Cover the role of mineral oil: prevent dehydration and concentration of droplets; make fertilized eggs in a sterile state; fix M2 solution droplets. Remove the fertilized egg from the incubator, aspirate the fertilized egg with a pipette and wash it with M2 solution 2-3 times, adjust the experimental dosage, place it in the M2 solution on the concave glass slide, and adjust the focus to make it clear under a low power See the outline of the fertilized egg, and ensure that the fertilized egg has enough space to move freely. Use the egg holder to gather the eggs together. When moving the egg, be careful not to move the bubbles into it, which affects the operating field of view. f. Microinjection equipment The basic working principle of the microinjection instrument is to use a stereo inverted phase contrast microscope for observation and monitoring. A micromanipulator is placed on each side of the microscope, one side is connected to the oviduct, and the other side is connected to the injection needle. The spatial position of the injection needle. The egg tube is connected to a syringe with fine adjustment filled with mineral oil through a plastic tube, and the movement of the egg is controlled by adjusting the pressure. The injection needle is connected to the injection instrument with a pressure pump through a plastic tube. After fixing the injection time and pressure, perform the injection operation. The operating system includes the LEICA AS TP gene transfer operating system (major instruments.co. Ltd) and the like, and the specific operating procedures are strictly carried out according to its instructions. 3) DNA loading in the injection needle Immerse the blunt tip of the injection needle in the tube holding the DNA to be injected, and the solution enters the injection needle through the siphon effect of the capillary pipette. The end of the injection needle should remain in the DNA solution until vesicles form at the end of the injection needle. This indicates that the DNA solution is completely loaded. Carefully inspect the end of the needle of the injection needle, a small concave surface can be seen a few millimeters away from it. Finally, the pipette filled with DNA solution can be installed in the needle holder or fixed in the instrument ring for use. 4) Microinjection of fertilized eggs The process of microinjection of fertilized eggs is relatively simple. In the process of making a large number of samples, in order to ensure the consistency of the amount of microinjection, a large number of repeated and effective exercises can be successful. a. Place the concave slide under the microscope and focus at low power. b. Adjust the egg-holding tube. After the injection needle and the fertilized egg are in the same visual field, the position is slightly lower than the fertilized egg when switching to the high magnification lens (32X), so that the fertilized egg can be operated freely. c. Close to the injection needle to the edge of the working fluid or oil boundary, and enter the oil boundary slightly. Before the injection, increase the pressure of the injection needle to see that the DNA solution bubbles in the oil to form a sac, to determine the existence of the DNA solution flow. d. If no DNA solution flows, gently rub the blunt edge of the sample holder and gradually open the syringe needle. The needle re-enters the oil to confirm the presence of the flow of DNA solution. e. Move the oviduct back to the lower part of the fertilized egg. By differentially driving the water pressure control system, a mild negative pressure is generated in the oviduct, and the end of the oviduct is sucked to the fertilized egg. This operation must ensure that the base of the fertilized egg base is in light contact with the base of the concave glass slide. Note that it should not be sucked too tightly, otherwise the egg will be deformed, and it will even suck the egg into the egg holding device. f. Slowly adjust the vacuum in the egg-holding tube, so that the fertilized egg in the egg-holding tube rotates gently, so that the pronucleus in the egg is located at the distal end of the egg-holding tube. g. Maintain the stability of the egg-holding tube, make the needle of the injection needle close to the transparent belt of the fertilized egg, adjust it so that the needle and the pronucleus are on the same plane. Use the injection needle to puncture the zona pellucida, the outer membrane of the cell, the prenuclear nuclear membrane, and enter the nuclear membrane. The zona pellucida of the fertilized egg is easy to be punctured by the needle tip. The anterior nuclear membrane is quite elastic. Try different methods to break through this structure. Avoid contact with the nucleus and damage the nucleolus during operation. h. Keeping the injection needle fixed, gently increase the pressure to allow the DNA solution to flow into the anterior nucleus. The possible phenomena during injection are as follows: â‘ After injection, the pronucleus will expand to about twice its original value, indicating successful injection, and then directly withdraw the injection needle. â‘¡ A bubble appears at the tip of the injection needle, and the zona pellucida may swell, indicating that the membrane of the fertilized egg is very difficult and has not been punctured. damaged. â‘¢ The pressure of the injection needle is high, and no phenomenon can be seen. The injection needle may be blocked. Needle replacement or DNA solution replacement is required. â‘£ If you see cytoplasm particles flowing into the space around the yolk, it means that the fertilized egg is broken. During the injection, if the number of ruptured eggs is large, the injection needle needs to be replaced. An injection needle can generally inject 5-10 eggs. i. Use the egg holder to move the fertilized egg to a relatively isolated position in the concave glass slide to distinguish the injection group from the non-injection group. Reinstall the oviduct and proceed to the next set of operations. 5. Tubal transfer of fertilized eggs 1) Tubal injection of fertilized eggs a. Place the anesthetized mouse on a plastic dish cover and fix the mouse teeth on the edge of the dish to ensure the airway of the mouse is unobstructed. Paint the incision site with 70% ethanol. You can also remove hair at the surgical site in advance. b. Transfer the fertilized egg from the culture fluid to the working fluid. Since the fertilized egg is operated outside the incubator during the transfer process, it should be removed from the medium to the working solution. c. Use a transfer tube to load the fertilized egg. The correct loading of the fallopian tubes is very important for the success of the fallopian tube transfer. As shown in Figure 11-1, draw a certain amount of working fluid at the tip of the oviduct, and then draw a little air to make a small bubble. Then suck up the working fluid about the volume of the bubble, and then suck another small bubble. Collect the fertilized eggs in the working fluid with the smallest possible volume and arrange them linearly in the oviduct. When all the eggs are loaded, draw a small amount of gas to make small bubbles, and then draw the final volume of working fluid. Air bubbles will help regulate the pressure and make it easier for eggs to move. d. Surgical exposure of the fallopian tube complex. As shown in the figure, make a transverse incision between the back hump and the hip joint about 0.5 cm from the midline. Carefully apply 70% ethanol to the incision site and remove hair. Pinch the skin on one side of the incision and bluntly separate the subcutaneous tissue. Move the skin until the abdominal wall nerves are clearly visible. At this time, red ovaries or light-colored ovarian fat pads under the abdominal wall can be seen. Squeeze the abdominal wall with ophthalmic forceps and make a lateral incision of about 0.5 cm to bluntly separate the tissue. Gently remove the fat pad, ovary, fallopian tube and uterus. Clamp the fat pad with a spring clip and keep the uterus in place. If the uterus and uterine horn frequently slide back to the abdominal cavity, the position of the uterine horn can be properly rearranged under the premise of ensuring the airway is unobstructed. e. Gently move the plastic dish to place the mouse under the dissecting microscope. Adjust the microscope and the mouse position to make the curled part of the fallopian tube clearly visible. f. Use ophthalmic forceps to bluntly open the small opening at the transparent capsule of the funnel, and prevent tearing of blood vessels, causing bleeding. It is necessary to apply epinephrine locally at the site of the tear to reduce bleeding, and wipe with gauze to keep the operating field clean. g. Once the funnel is clearly visible, use tweezers to grip its edge and fully expose the funnel nozzle. Under the premise of avoiding ampulla injury, insert the fallopian tube as much as possible. h. Under the premise of adjustable pressure, gently blow the egg into the funnel. Air bubbles can prevent the egg from flowing back and it is easy for the egg to enter the fallopian tube funnel. If the pressure of blowing the egg is too high, the mouth of the fallopian tube may be against the wall of the fallopian tube. At this time, the fallopian tube may be withdrawn slightly for operation, or the transfer tube may be blocked by a blood clot. In the petri dish, suck the eggs again. i. After the egg transfer operation is completed, withdraw the oviduct, remove the instrument, and reset each organ in the abdominal cavity according to the original position. j. Suture the incision and hold the skin with a small clip. Automatic small clips are often used instead of sutures, which can prevent mice from gnashing sutures and splitting the incision. k. If performing bilateral surgery, repeat the above procedure on the other uterine horn. l. After the operation, place the mice in a clean cage. Under anesthesia, small mammals cannot effectively maintain body temperature. Therefore, attention should be paid to the heat preservation of mice. A hot pad can be used to maintain its temperature until the animal wakes up. All animals can wake up 20-30 minutes before returning. Because pregnancy can easily cause stress in the recipient female rat and result in miscarriage or feeding, the recipient female rat must be strictly monitored. 2) Post-injection monitoring: It is very important to strictly monitor and prevent complications after surgery. Anesthesia easily induces increased blood pressure in mice, so it must be closely monitored for at least 2 hours after surgery, and heat preservation treatment is recommended. Mice under anesthesia should be wrapped with soft gauze, and straw mats and soft materials should be added to the cage, and the temperature of the cage should be kept. The maintenance of normal body temperature can shorten the time the animal is under anesthesia. After the operation, the mice are observed once every 4-5 days to ensure that the mice are recovering, and the conscious mice should be free to move. After abdominal surgery, mice may develop intestinal hernia. Therefore, the incision should be kept as small as possible during the operation, and the suture should be tight. The correct application of tissue glue can avoid such complications. The skin must be closed with sutures or stainless steel clips, which can be removed 1-2 weeks after surgery. If the animal is in poor condition, exhibits anorexia, dehydration, or obvious bow back, hydroxyphenylacetamide and similar analgesics can be given through the animal's water. If dehydration occurs, 0.9% saline or Ringer's solution can be injected intraperitoneally. If there is still no improvement, reopen the hand incision under anesthesia to confirm whether there is a hernia. If there is no obvious improvement in the animal's condition, euthanasia will be used. 5. Identification of transgenic mice Among the mice produced, they are transgenic mice, accounting for about 20% -30% of all the mice. Therefore, identification and screening of transgenic mice is necessary. 1) Transgenic integration testing The easiest way to identify transgenic mice is to extract genomic DNA from the tail tip of the mouse and detect its genotype. Detection methods include PCR and Shouthern hybridization. a. Extraction of genomic DNA: Weaning mice (> 4 weeks old) are anesthetized. Grab the mouse with one hand and cut the mouse tail approximately 1 cm with the other hand holding a sterile scissors. Put the cut rat tail in 500 μl of digestion buffer (50mmol / L Tris-HCl, pH8.0; 100mmol / LEDTA; 100mmol / LNaCl; 1% SDS), and add proteinase K to make the final concentration 100 ug / ml, shake at 55 ℃ for 3 ~ 4 hours or overnight. Add 5 ul RNase A and incubate at 370C for 1-2 hours. b. PCR detection: The initial screening of transgenes usually uses PCR detection technology. The technique is simple, fast, low-cost and effective, and is suitable for the analysis of a large number of specimens. Because this technique is particularly sensitive, it may produce false positive results. Therefore, special care must be taken during the operation to avoid contamination of plasmid DNA or genomic DNA of other specimens. The generation of false positives will be fatal to the screening of transgenic mice. PCR experiments should use double tubes, even triple tubes. The positive result is best confirmed by Southern hybridization technique. c. Southern blot analysis: Although this technique is not as sensitive and time-consuming as the PCR technique, it avoids the trouble of false positive results due to contamination, and can obtain the integrated genome of the target gene, the number of integration sites, the number of transgenic copies, etc. Exact information. 2) Transgene expression detection Transgene integration testing is to determine whether the target gene is integrated into the mouse genome, and at the same time to determine the integration site and copy number, which is very important in genetics. The detection of transgene expression is to determine the spatial and temporal distribution of the expression of the target gene in the organs and tissues of transgenic mice. Its detection includes RNA analysis technology and protein detection technology. a. RNA isolation: Extract total RNA or mRNA from transgenic mouse tissues or cells according to the needs of the experimenter. Isolation of total RNA is relatively simple and suitable for gene transcription analysis. b. Northern blot analysis: This technique is used to qualitatively detect the relative level of transgenic transcription in transgenic animal tissues or cells. c. RT-PCR detection: This technique can quantitatively or semi-quantitatively detect the mRNA specifically expressed by transgenes in transgenic mouse tissues or cells, and is very sensitive. Transcripts that cannot be detected by Northern blotting can also be detected by this technique, One copy of transcripts in 1000 cells can be detected. d. Western blot analysis: This technique is usually used for the expression level of transgenic protein in transgenic mouse tissues or cells. e. Immunohistochemical analysis: This method can detect the tissue distribution of transgenic protein expression in transgenic mice. There are a variety of experimental methods, you can refer to the relevant immunology detection technology books. 2 Person Sound Proof Meeting Pod 2 Person Sound Proof Meeting Pod,soundproof pods office,office phone booth Feat Top International(China) CO.,LTD , https://www.feattop.com