转基因小鼠F0 z-bo 2020-10-14 514

　　1. Preparation of DNA for microinjection of prokaryotes: usually linearized and purified by electrophoresis, gel recovery, precipitation and purification. The working concentration is usually 1-5g/μl. Care should be taken because the purity and concentration of DNA here will have a major impact on subsequent success rates.

　　2. At the same time, donor female mice (produced by the mating of hormone-injected female mice and normal male mice) and pseudopregnant female mice (produced by the mating of normal female mice and ligated male mice) are obtained. Donor mice are used to collect fertilized eggs for injection, and pseudo-pregnant female mice are used to survive fertilized eggs after injection, and finally F0 generation transgenic mice are produced.

　　3. After injecting the gonadotropin of the villi into the donor mice, collect pronuclear fertilized eggs with clear nuclei. Chorionic gonadotropin can make female mice ovulate twice as often as normal ovulation. Fertilized eggs for microinjection are best collected in the morning after mating and several hours before injection. At this time, the injection operation is easy.

　　4. Using microinjection, the purified foreign gene is injected into the pronucleus of the fertilized egg. The fertilized eggs in the working fluid will form clumps, and the fertilized eggs used for microinjection must be single cells without cell debris. If it does not meet this standard, it must be digested with hyaluronidase and then rinsed.

　　5. Identify fertilized eggs, surviving fertilized eggs or surviving embryos, and transfer them to the Farrobius tube (or uterus) of pseudo-pregnant female mice that are synchronously mated, regardless of whether they are pregnant or not, and determine after individual reproduction State of development.

　　6. Detection of foreign gene integration and expression in offspring mice. Transgenic mice are among the puppies produced, accounting for about 20% to 30% of all mice. Therefore, there is a need to identify and screen transgenic mice. The most common methods to identify whether genes are integrated are PCR and Southern hybridization. Orthern hybridization, RT-PCR detection, Western blot and immunohistochemistry are used to detect the protein expression status of transferred genes.

　　7. Establish a strain of transgenic mice.

　　In the above part, in addition to microinjection, we also designed many sophisticated operations, such as ligating male mice, separating fertilized eggs from mothers, and conducting animal experiments, such as transplanting fertilized eggs. The process is cumbersome, the preparation of the experiment is very careful, and it is also necessary to understand the principles and operations of animal physiology, anatomy and molecular biology. It is difficult to establish a stable and efficient platform in a short time.