The development of transgenic technology is remarkable. Among them, the achievements of transgenic technology of plants and microorganisms have been industrialized, bringing huge economic benefits to mankind. Transgenic animals, especially transgenic poultry, have become a hot topic at home and abroad due to their superior production capacity and good market prospects. First, the production of transgenic birds 1. Microinjection of transgenic chickens (l) single cell fertilized egg microinjection method. It was found that when exogenous DNA is injected into the cytoplasm of fertilized eggs of vertebrates such as sea urchins, zebrafish, and Xenopus, when the eggs rapidly divide, exogenous DNA temporarily stays extrachromosomally replicable and can be incorporated into the chromosomal genome at a low frequency. . Thanks to the enlightenment of the above studies, the exogenous gene was injected into the unicellular fertilized egg cytoplasm in vitro. After in vitro culture, the transgenic chicken was successfully produced. However, if a fertilized egg is used to obtain a hen in this method, if the operation fails, the loss of one cell is equivalent to the loss of a hen, the cost is high, and the in vitro hatching condition of the fertilized egg is also very complicated, and the operation difficulty is difficult. Larger. (2) Pronuclear injection of fertilized eggs. In China, Li Zandong et al. injected liposomal packaging plasmid pMiwz (containing reporter gene LacZ) into the blastocyst stage blastocyst, and successfully obtained the transgene, and proved that transfected primordial germ cells can be obtained by injecting exogenous genes at the blastocyst stage ( PGCs) and can be passed on to the next generation. In foreign countries, Jamie love et al. injected the plasmid DNA with the target gene into embryos of chicken fertilized eggs. After 12 h of in vitro culture, nearly half of the embryos were found to survive, 40% of the embryos contained plasmid DNA, and 6% corresponded to 1 cell per cell. The copied exogenous gene, one of seven surviving chicks of sexual maturity, passed 3.4% of the exogenous gene to its offspring. This method can obtain transgenic chimeric chickens, but the mosaic site and degree of mosaic of the transgenic cells are not easy to determine. 2. Trajectory transfection of embryonic embryonic chicken embryos This technique was originally applied to the genetic manipulation of plant cells. It includes two major components: the ion acceleration system and the ion packaging system. This method is more efficient, and since tungsten ions of different sizes are used, and the embryonic new moon area is mainly yolk protein, it is not necessary to consider the velocity of the projectile and the penetration depth (Bai Zhantao et al., 2000). However, because the DNA introduced by this method is difficult to integrate into the chicken's genome, genetic stability and passaging are not strong. 3. Retroviral vector method This method uses the virus's normal life cycle characteristics to infect poultry, thereby introducing foreign and achieving the purpose of integration. There are two types of viral vectors used today: replication-complete and replication-defective (Haopo, 2000). A replication-competent retrovirus contains all structural genes that are self-replicating and can infect cells repeatedly. The replication-defective retrovirus lacks some or all of the structural genes and cannot self-replicate and needs to be propagated in helper cells. The integration efficiency of reverse transcription vector method is higher than that of electroporation and lipofection, but due to the restriction of the size of the gene fragment and the difficulty in obtaining high-titer virus, it has mainly stayed in the laboratory and has not been widely used. Commercial production (Liu Xiangping, 2003). 4. Sperm carrier method This method utilizes the normal physiological process of sperm-egg binding to complete the introduction of foreign genes. It is simple and easy to perform, and has no damage to the pronucleus of eggs. However, the damage to sperm is a very thorny problem, and the integration The degree is not high. (1) Liposome-mediated sperm vector method. In China, Du Lixin and others have discussed this method. The general experimental steps include preparation of liposome-DNA complexes, artificial sperm insemination with mDm-His method, and detection of transgenic chickens. The problem was found to be a decrease in sperm motility and a decrease in the expression rate of exogenous genes with embryonic development. (2) Sperm cell electroporation. Electroporation is an important method to promote the binding of DNA and animal sperm. It uses a high-voltage electric field to create temporary holes in the plasma membrane of the sperm, making it easier for foreign DNA to enter the cell. According to Zhong Jiayu's research, the dehydration and rehydration of spermatozoa or the electroporation of sperm in a bath in an exogenous gene are all performed. The positive rate obtained is much greater than the mere mixing of the exogenous gene and sperm. First dehydration and then placed in the hypotonic fluid, the sperm will inhale the hypotonic fluid, and it is this process to greatly increase the positive rate, the foreign gene is very likely to enter the sperm with hypotonic fluid. 5. Embryonic stem cell primordial germ cell manipulation embryonic stem cell (ES) is a pluripotent cell line that has been established in vitro from the inner cell mass of early embryos. When ES is injected into stage X blastocysts, it can participate in the formation of various tissue chimeras including gonads. Usually, foreign genes are introduced into ES cells using retrovirus vectors, electroporation methods, etc. Genes are integrated into non-essential gene loci in the ES cell genome, screened, cultured, and then used for transgenic chicken production. Second, the application of genetically modified chicken Genetically modified chicken research in animal breeding, bio-pharmaceutical applications in specific values ​​in the following aspects: 1. Accelerate the process of QTL selection In some circumstances, transgenic poultry can be used as a candidate for verification of quantitative traits A method of site hypothesis. For example, we have identified a QTL for a certain trait from a whole group of chicken genomes using a molecular marker. We can then mutate these sites in sequence. Then one by one according to the phenotype exclusion, the final determination of major genes. However, a single gene that is mutated or has a large effect inserted into the genome may produce a series of unfavorable phenotypic responses, as it often appears when selecting a single trait under high selective intensity, which may give the research process Inconvenience, such as high mortality rate, maladjustment of overall function, etc., can affect the performance of traits (Yan Huaxiang, 1994). However, with the deepening of genome function research, many negative performances will be overcome, and transgenic methods may replace traditional selection methods. 2. Improve the yield and quality of meat eggs. Genes can be introduced into the genome of chickens through genetically modified hormone and growth factor genes, or some genes with unfavorable production performance can be eliminated, thereby greatly increasing their yield. For example, the cellulase gene in ruminant intestinal cells is introduced into the chicken body so that the chicken body can decompose the polysaccharide. Thus, herbivorous and long-fleshed chickens are produced, and the feed source of the chicken is increased. 3. Perform disease-resistant breeding application of gene knock-out technology to destroy chicken's endogenous virus receptor gene, which can prevent virus invasion. Marker-assisted selection techniques can also be used to identify sites related to disease resistance in chickens, followed by PCR amplification, and then introduced into chicken embryo cells by appropriate methods, and the homologous sequences can be replaced using enzyme digestion techniques. Embryonic cultures are then performed, and disease-resistant strains are finally cultivated in conjunction with quantitative genetics. 4. After the production of medical and health proteins followed by mammary gland bioreactors, transgenic chicken bioreactors have become a new hot spot. Through the introduction of genetically engineered genes for various human and animal antibodies to prevent disease in humans and animals, SOD gene and human leptin protein genes can be introduced into the genome of chickens to produce cosmetics and health products. Because genetically modified chickens have their special advantages, the research on the use of genetically modified chickens for the production of drugs has received great attention and attention in the world and will inevitably produce great economic and social benefits. "World Agriculture" 2004 Second Period Wang Xiaotong Yan Yizhou Wang Xiaona Authors: College of Animal Science and Technology, Yunnan Agricultural University Zhaoyuan City Animal Husbandry Veterinary Station, Shandong Province
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