Gene animal refers to a kind of animal that stably integrates foreign genes into chromosome genes and can be introduced into stable expression through experiments. From 65438 to 0974, Jaenisch successfully obtained SV40DNA transgenic mice by microinjection for the first time in the world. After that, costantini injected rabbit globin gene into mouse fertilized eggs, so that the fertilized eggs developed into mice and expressed rabbit globin. Palmiter et al. introduced rat growth hormone gene into mouse fertilized eggs to obtain "super" mice; Church got the first transgenic cow. So far, people have successfully obtained transgenic mice, chickens, goats, pigs, sheep, cows, frogs and a variety of transgenic fish.
1. Transgenic animal technology
Prokaryotic microinjection 1. 1
Also known as DNA microinjection, that is, the foreign gene is directly injected into the fertilized egg with a syringe through a micromanipulator, and the foreign gene is integrated into DNA to develop into a transgenic animal. Its founders are Jaenisch and Mintz. Gordon et al. and Palmiter et al. successively obtained transgenic animals by this method. This method is widely used at present, and the research on transgenic animals is mostly based on Palmiter and other methods. Wang (1996) reported that the nuclease gene against plague virus was introduced into rabbits by microinjection to obtain transgenic rabbits. KrimPenfort obtained transgenic cattle by in vitro embryo culture and microinjection.
This method is characterized by high efficiency of foreign gene introduction and integration, direct transfer of target gene without vector, and the length of target gene can reach.
1.2 transcription virus vector method
Recombination of the target gene into a retroviral vector, making high-concentration virus particles, artificially infecting the embryo before or after implantation, or directly incubating the embryo with a monolayer culture cell capable of releasing retrovirus to achieve the purpose of infection, and inserting the foreign target gene into the host genome DNA through the virus. In this way, Slater and others got transgenic chickens and haskell got transgenic cattle.
Retrovirus transformed by recombinant DNA technology is superior to microinjection in application: it does not need rearrangement, and a single copy of the transferred gene can be integrated at the integration point; When the embryo is placed in a high concentration virus container, or co-cultured with infected cells in vitro, or microinjected into chicken blastoderm, the embryo rate of integrating retrovirus DNA is higher. The disadvantages are: it is necessary to produce retrovirus with transgene; There is a certain size limit for the gene inserted into retrovirus; The obtained transgenic livestock has high chimerism and needs a lot of hybridization to establish transgenic strains; The problem of transgenic expression has not been solved.
1.3 Embryonic stem cells (ES cells for short) introduce genes into embryos; Then, after cells are injected into animal blastocysts, transgenic embryos can participate in the embryonic formation of the host and form chimeras until species chimeras are reached. Therefore, it can be used as a vector to introduce foreign genes and obtain transgenic animals. That is, a pluripotent cell line established by in vitro culture from the inner cell mass of early embryos.
Its advantages are that before transplanting embryonic stem cells into embryos, specific genotypes can be selected in vitro; After exogenous DNA transfection, embryos can be cloned in cells, and then cells containing integrated exogenous DNA can be screened for cell fusion, so that many genetically identical transgenic animals can be obtained. The disadvantage is that many chimera transgenic animals do not contain transgenes in their germ cells;
At present, the application of embryonic cell-mediated method in mice is mature, but it is late in large animals. Evans et al. (198 1) used different culture systems to isolate and establish pluripotent stem cell clones from the inner cell mass of mouse blastocysts. Stice and
Strelchenko et al. (1996) obtained bovine embryonic stem cells.