胚胎干细胞法 z-bo 2020-10-14 368

　　The Martin Evans Institute has successfully cultivated a pluripotent stem cell line from the internal cell mass of early embryos, which can grow in vitro and can differentiate into other cells even if it is reinjected into germ cysts These cells (including germ cells) are called embryonic stem cells (embryonic stem cells, ES cells). ES cells can be cultured in vitro for a long time to maintain an undifferentiated state, and to modify and screen their genomes.

　　Next, the obtained ES cell clones are injected into the scutellum sac, and the ES cells are integrated into the early embryo to participate in embryonic development and various tissues and organs. If it participates in the development of the testis and forms sperm derived from ES cells, the genetic changes can be passed on to the next generation to obtain knockout or transgenic animals. The first transgenic animals produced in ES cells were transgenic mice infected with retroviral vectors. ES cells are mainly used for gene targeting to generate knockout mice. Transgenes smaller than 50 kb usually do not require ES cell methods to produce transgenic animals, but ES cell pathways are large genes (50-400 kb) or very large genes (50-400 kb) or very large genes that are very difficult to microinject gene. Especially important for 400kb-3Mb).

　　"For example, large genes such as TCR and Ig genes can be cloned into YAC vectors with 800-3000 kb sites, and the preparation of transgenic mice usually requires the fusion of ES cells and YAC cells. Es-yeast cell fusion method: This technology is an important method for preparing super-large gene transgenic animals and many super-large fragment YAC DNA transgenic animals, such as immunoglobulin and TCR locus transgenic mice. Used to make mice. This method does not require purification of YAC DNA. There is no obvious limit to the size of DNA. You can transfer 500-2000 kb DNA by fusion method. Yeast cell membrane has a hard cell wall. After digesting the cell wall with enzymes, spheroplasts are formed, which are fused with mammalian cells (mouse ES cells, etc.) by PEG fusion method to introduce YAC gene into animal cells. I can. Yeast DNA and YAC DNA can be transferred into mouse ES cell genome at the same time. The transfer efficiency of large intact YAC DNA fragments to the ES cell genome is very high, and the proportion of ES cell clones with intact YAC DNA is close to 95%. When YACDNA is transferred, some or all of the yeast chromosomes are also transferred to the mouse genome. The yeast genome is usually not expressed in mammalian cells and does not affect the pluripotency of ES cells or mouse embryonic development.

　　You can introduce Transgene. Next generation. Recently, by reprogramming cells with transcription factors Oct4, Sox2, Klf4, and c-Myc, it has been possible to induce mouse embryonic fibroblasts to produce pluripotent stem cells (iP). .. Through a process called tetraploid cyst complementation, iPS cells have been used to produce live mice. Although still at its own stage of development, this technology has the potential to provide a powerful way to produce transgenic animals from transgenic embryonic fibroblasts in vitro. ES cells are considered as new test materials in research fields such as transgenic animals, nuclear transfer and gene therapy, and have a wide range of potential applications. However, ES cells have not been isolated from animals such as cattle, sheep, pigs and chickens. Currently, the use of ES cells to produce transgenic animals is limited by two conditions, and better methods can be used instead. First, only mouse ES cells are currently commercially available, and ES cells from other animals have been reported. For example, rat ES cells have been successfully established to generate P53 knockout rats. Secondly. Even in ES cells, chimeras must reproduce during reproduction, so this is not a big problem for mice. When it is applied to other livestock with long breeding cycles and high raising costs, certain problems will arise. Of course, ES cells are excellent nuclear donors and are easy to use for storage and animal cloning.