GB/T 14927.2-2001 Laboratory Animals - Skin Transplantation Method for Inbred Mice and Rats
Some standard content:
National Standard of the People's Republic of China
GB/T14927.2—2001
Laboratory animal
Genetic monitoring: skin grafting of inbred mice and rats2001-08-29Promulgated
Implementation on 2002-05-01
People's Republic of China
General Administration of Quality Supervision, Inspection and Quarantine
GB/T14927.2—2001
This standard amends the contents of GB/T14927.2—1994 "Standard for equipment, materials and operating procedures for laboratory animals. Skin grafting of inbred mice and rats" and is a revision of the original standard. This method is a classic method for purity detection of inbred mice and rats with a small inbred strain, a long application time and simple operating technology. There are no further changes in this revision.
This standard shall replace GB/T14927.2-1994 from the date of implementation. This standard is proposed and managed by the Ministry of Science and Technology of the People's Republic of China. The drafting unit of this standard: China Laboratory Animal Society. The main drafters of this standard: Xing Ruichang, Liu Shuanghuan, Shen Jie, Li Shanru, Bai Qinhua, Zhang Ruizhong This standard was first issued in January 1994.
National Standard of the People's Republic of China
Laboratory Animals
Skin transplantation of inbred mice and rats
Laboratory animal-
Genetic monitoring: skin transplantation of inbred mice and rats Scope
This standard specifies the skin transplantation of inbred mice and rats. GB/T14927.2—2001
Replaces GB/T14927.2—1994
This standard applies to the purity inspection of inbred mice and rats during the breeding process and the genetic monitoring of inbred mice and rats during the breeding and feeding process.
Technical principle
The transplanted tissues can be mutually accepted in the same inbred strain, that is, isograft is successful. The transplanted tissues are mutually rejected in different inbred strains, that is, allograft is unsuccessful. F1 generation animals can accept tissue transplants from any parent, but the parents cannot accept transplants from F1 generation. F1 generation animals can accept transplants from F2 generation and later generations. Parent strains can accept some transplants from F2 generation and later generations, but most of them are rejected. This standard adopts the back skin transplantation method and the tail skin transplantation method. The two methods have the same principle and have the same standard effect. Back skin transplantation method
Equipment and materials
Fixation board (18cm×12cm).
Nembutal sodium (medical).
Medical adhesive plaster.
3.1.4Medical vaseline.
3.1.5Powdered penicillin G sodium (800,000 U, for human or animal use). 3.1.63% iodine cotton balls.
3.1.775% alcohol cotton balls.
3.1.8Ophthalmic scissors.
3.1.9Ophthalmic forceps.
Syringe (1mL).
No. 4 or No. 5 syringe needle.
Gauze (cut into 40mm long, 25mm wide strips: 2-3 layers thick, several pieces, coated with medical vaseline and powdered penicillin G sodium).
3.1.13 Cotton balls made of absorbent cotton.
3.1.14 Place the materials in 3.1.10 and 3.1.13 in a pressure cooker and sterilize them at 121℃ for 40min. 3.2 Operation steps
3.2.1 Randomly select 10 animals of the same sex aged 4 to 12 weeks. The animals can come from the basic group, blood expansion group or production group. 3.2.2 Number and weigh each animal separately, and record the name of the strain, sex, date of birth, pedigree and other characteristics in detail. 3.2.3 Prepare 0.7% sodium pentobarbital solution with sterile saline. 3.2.4 Anesthetize the animal by intraperitoneal injection. Rats are injected with 0.1mL for every 20g of body weight, and mice are injected with 0.1mL for every 10g of body weight. Different strains of animals have different sensitivities to anesthetics. The injection volume can be appropriately increased or decreased (the room temperature should be controlled between 25℃ and 28℃ during surgery). 3.2.5 After the animal is anesthetized and loses consciousness, place it on a fixed board with its back facing upwards, fix the animal, cut off the hair, and disinfect it with 3% iodine cotton balls and 75% alcohol cotton balls.
3.2.6 Cut a piece of skin with a diameter of 5mm to 10mm on the back (one piece is used for autologous transplantation and the other piece is used for allogeneic transplantation).
3.2.7 Turn the cut skin over and put it in a double dish (diameter = 6cm) with a small amount of saline. Use ophthalmic scissors to gently cut the subcutaneous tissue to the dermis, and then rinse it in sterile saline. 3.2.8 The skin of the two animals, except for the left skin for autologous transplantation, the right skin is exchanged in a loop, transplanted against the direction of hair and anastomosed. 3.2.9 Cover with a gauze piece coated with vaseline and penicillin G sodium, 34 layers, fixed with 1cm wide adhesive tape, with appropriate tightness. 3.2.10 After the operation, when the animal wakes up, put the animal in a mouse box and hang a marking card. Remove the bandage after 10 days. 3.3 Result observation
3.3.1 After unpacking, if the skin graft is dry, painful, and falls off, it is a technical failure. If the skin graft falls off, the surgical site is flat, and new hair grows after one week, the operation is successful. Compared with autologous transplantation, the technical failure rate shall not be greater than 10%. 3.3.2 If the skin graft falls off within 2 to 3 weeks, it is acute rejection. Genetic contamination usually causes acute rejection. 3.3.3 If the skin graft falls off within 3 weeks, it is chronic rejection. Whether the graft has chronic rejection should be observed for at least 100 days. Genetic mutations usually cause chronic rejection.
3.3.4 If there is doubt about the result, a new transplant should be performed. A new batch of animals can be used, or animals that have been transplanted but have doubts about the results can be used. If it is the latter, the rejection is faster and more typical. 4 Tail skin transplantation method
4.1 Equipment and materials
4.1.1 No. 11 surgical knife handle.
4.1.2 No. 11 surgical blade.
4.1.3 Glass sleeve (8mm diameter, larger for rats). 4.1.4 Other materials see 3.1.
4.2 Operation steps
4.2.1 Randomly select 10 animals of the same sex aged 4 to 12 weeks. The animals can be from the basic group, blood expansion group or production group. 4.2.2 Number and weigh each animal separately, and record the strain name, gender, date of birth, pedigree and other characteristics in detail. 4.2.3 Prepare 0.7% sodium pentobarbital solution with sterile saline. 4.2.4 Use intraperitoneal injection to anesthetize the animal. Rats are injected with 0.1mL for every 20g body weight, and mice are injected with 0.1mL for every 10g body weight. Different strains of animals have different sensitivities to anesthetics. The injection volume can be appropriately increased or decreased (the room temperature during surgery should be controlled between 25℃ and 28℃). 4.2.5 After the animals are anesthetized and lose consciousness, place them in a supine position on a piece of filter paper in groups of 5 in order, and disinfect the tails with 3% iodine cotton balls and 75% alcohol cotton balls.
4.2.6 Press the base of the animal's tail with the index finger of the left hand and the tip of the tail with the thumb of the left hand, fix the tail and stretch it slightly, then hold the scalpel in the right hand, with the blade facing up, at an angle of 20° to 30° with the tail skin, and cut off a piece of skin about 2mm to 3mm wide and 7mm to 8mm long at the upper part of the tail vein or between the two tail veins, 5mm away from the tail. The thickness of the former should be such that there is no severe bleeding, and the latter should be enough to expose the white tendon but not cut the blood vessels. 4.2.7 The right hand passes the blade to the left hand in a counterclockwise direction. The skin attached to the blade is rotated 180° accordingly. The skin is removed with ophthalmic forceps and attached to the original surface, and it is matched as much as possible. It is covered with a small piece of filter paper, and then gently pressed. Then the filter paper is removed. This transplantation is used as an autologous transplantation control.
4.2.8 According to steps 4.2.6 and 4.2.7, the autologous transplantation of another 4 transplantation control
animals
4.2.9 According to steps 4.2.6 and 4.2.7
, the third skin and the second 1
of the adjacent mouse behind
are used for circular skin transplantation. That is, the specific operation of the front mouse is shown in Figure 1.
A-Autologous transplantation control: 1, 2, 3; 4, 5-Numbered mouse donor skin grafts Figure 1
Skin transplantation mutual circulation system
4.2.10 Take five glass cannulas and gently insert them into the animal's tail to 3mm from the root. Use medical tape to stick the tail at the far end of the tail near the cannula. Stick the tape back and forth 2 to 3 times so that the cannula can move slightly up and down but not fall off. 4.2.11After putting on the glass tube, use a floor-standing surgical lamp to irradiate the animal for about 15min to 30min. Then put the animal in a mouse box with a marking card on its back.
4.2.12After 24 hours, remove the tube. At this time, you can see that the skin graft has adhered to the wound surface. 4.3 Observation of results
4.3.1 If the skin graft becomes pale, dry, or falls off within the first week, it is a technical failure. Compared with autologous transplantation, the technical failure rate shall not be less than 10%. 4.3.2 If the skin graft becomes inflamed, edematous, necrotic, or scaly until it falls off within the 2nd to 3rd week, it is acute rejection. Genetic contamination usually causes acute rejection. bZxz.net
4.3.3 If the hair on the skin graft gradually falls off within the 3rd to 9th week until it is hairless; or if a sunken scar is left due to rejection, it is chronic rejection. Genetic mutations usually cause chronic rejection.
4.3.4 If the skin graft has reverse hair growth during the 100-day observation period, it is a sign of permanent acceptance. 4.3.5 If there is any doubt about the result, re-transplantation should be performed to obtain a clear result.
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