Preparation of transgenic mice
Preparation of transgenic mice
The transgenic mouse preparation experiments can be applied to (1) animal developmental studies; (2) study of cell function regulation mechanisms.
Operation method
Preparation of transgenic mice
Principle
The basic material of heredity is DNA, and genes are DNA segments located on chromosomes with hereditary effects. For all the genetic information stored in the complete set of chromosomes of organisms, it can be called the genome. The genetic composition of different kinds of organisms and different individuals is different. For individual animals, the genetic components that are not their own belong to exogenous genes, if the exogenous genes are integrated or imported into the genes of the animal chromosomes, the exogenous genes are known as transgenes (i.e., transferred genes), and this kind of animal is a transgenic animal.
Materials and Instruments
Mouse Move 1. 7~8 weeks old female mice with closed vaginal opening were selected as donors and each mouse was injected intraperitoneally with PMSG (10 IU) at around 3:00 pm. Caveat 1. Keep the tip of the injection needle away from personnel and instruments in the laboratory throughout the procedure. When the injection needle is not securely mounted on the needle holder, the pressure inside the syringe, tubing and injection needle may eject the needle. 2. Injecting too fast can disturb cytoplasmic components, cell lysis or cell displacement from the substrate. Injections work best when the volume injected is less than 50% of the estimated cell volume and when the flow rate of the injected sample causes little or no visible cytoplasmic displacement. 3. Live cells can be visualized with UV light after injection of markers, but irreversible damage to the cells can occur, so UV exposure should be kept as short as possible. Common Problems 1. The optimal components vary significantly depending on the particular cell type used. If efforts to optimize voltage and pulse width for electroporation are unsatisfactory, attempts should be made to change the perforation medium. 2. Another important factor affecting electroporation/electrofusion is related to the cellular state; for maximum efficiency, it is necessary to collect cells at mid-logarithmic growth. For more product details, please visit Aladdin Scientific website.
HCG PMSG Hyaluronidase Sodium pentobarbital
Microscope Tweezers Egg-holding needle Injection needle
2. 47~48 hours later, each mouse was injected intraperitoneally with HCG (0.8 IU) and caged with normal male rats; several female rats of the appropriate age (2 months old or above) were also taken as recipients, with a flushed vaginal opening, and caged with ligated male rats.
3. Observe donors and recipients before 9:00 a.m. on the second day and take out those with sperm plugs. Take out the receptor cages for isolation measures.
4. At around 10:30, the donor was executed by neck-breaking, and the entire fallopian tube was surgically removed and placed in hyaluronidase ~0.3 mg/M2 solution. Under the microscope, the pot-belly of the fallopian tube was torn open with forceps, and the fertilized egg, along with the granulosa cells, flowed out.
5. Carefully observe the fertilized eggs placed in hyaluronidase M2 solution, when the granulocytes around the fertilized eggs detach, aspirate the fertilized eggs, put them into M2 solution for washing, and finally put them into M16 solution in 5% CO2 and incubate them at 37℃.
6. Observe under a microscope and select fertilized eggs with full cells, clear zona pellucida and clearly visible male prokaryotes for use.
7. Attach the needle and syringe so that the end is parallel to the carrier, place a drop of M2 in the center of the concave slide, cover with paraffin oil, and transfer the fertilized egg to be injected. any air bubbles of DNA in the syringe should have been popped earlier.
8. Under high magnification microscope, touch the injection needle gently to the oviduct to make DNA flow out slowly and control its flow; blow the fertilized egg repeatedly to make it in the best position, and insert the injection needle into the male nucleus of the fertilized egg until the nucleus expands, then withdraw. The injected and uninjected fertilized eggs were placed separately at the top and bottom to avoid mixing, and after injection, they were put into 5% CO2 and incubated at 37℃.
9. The recipient is anesthetized and injected with a measured amount of 1% sodium pentobarbital 0.01 ml/g intraperitoneally. The ovaries were surgically removed to connect the fallopian tubes, which were secured with fat forceps and the tubal openings were located under the microscope. Fertilized eggs that were cultured and viable after injection were aspirated by aspirating a longer section of M2, aspirating one bubble, then aspirating the fertilized eggs, as closely aligned as possible, then aspirating a section of fluid, aspirating one bubble, and then aspirating another section of fluid, for a total of four sections of fluid and three bubbles. Except for the longer section of fluid, the rest of the fluid was roughly 1 cm and the bubbles 0.2 cm. Insert the port of the explant tube into the mouth of the fallopian tube and gently blow the fluid into the explant tube. The explant is successful when the potbelly of the fallopian tube is seen to expand and three air bubbles are clearly visible. The ovary was put back into the abdominal cavity together with the fallopian tube, and the muscle and skin were sutured.
10. Recipients are weighed every other week, and pregnancy is initially recognized when the second weighing increases over the first. The pups were delivered 19-21 days after surgery, and after the pups were 3 weeks old, the ears were clipped and numbered, the tails were clipped, and the pups were submitted to the molecular group for testing.
(Female mice of 4-5 weeks of age are usually selected as donors, when the mice have more eggs and are in better condition. Oocyte maturation was induced with pms and superovulated with hcg.)