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2 Stem cell-mediated method Embryonic stem cell-mediated method has also emerged in the early 1980s for the weakness of microinjection. This method involves the isolation of embryonic stem cells from embryos, introduction of foreign genes into the cells by transcription, and transfer into the blastocyst space of preimplantation embryos. Embryonic stem cells (ES cells) with foreign genes can be inserted. The inner cell mass of the host blastocyst is involved in embryonic development. The reproductive system of a future-born animal may be integrated with an exogenous gene, and an individual having a homozygous target gene, ie, a transgenic animal, may be obtained through cross breeding. This method is simple and easy, and the integration rate is also high. At the same time, the expression, integration, and stabilization of foreign genes can be selected at the cellular level (fertilized eggs), which reduces the workload and shortens the work cycle. However, the transgenic animal produced by this method is a chimera, which brings certain difficulties to practical application.
3 retrovirus method appeared in the late 1980s retrovirus method, retrovirus is a type of RNA replication through the DNA virus, after infection of the host cell, the virus genome by reverse transcription into DNA, known as provirus DNA, one of the proviral DNAs can be integrated into the chromosome of the host cell, and the exogenous gene is linked to the viral RNA by molecular techniques. The virus is infected by the proliferation of the virus itself, and the carried gene is transferred to the recipient cell. The foreign gene is integrated into the The host chromosomes are expressed in mature individuals. Compared with the microinjection method, the retrovirus method has the advantage of being able to integrate at various stages of the embryo, and the integration position is specific, does not lead to the rearrangement of the host genome; this method is simple and does not require special equipment. However, when retroviruses infect a host, they are non-homologous, structurally unstable, and interactions occur in the regulatory regions, and the length of the exogenous gene fragment is limited, and cell replication is also required. Therefore, the retrovirus method requires certain characteristics of foreign genes and retroviruses. However, the retrovirus method is currently the most efficient method of producing transgenic chickens.
Transgenic Technology 3
1 Microinjection In many transgenic technologies, microinjection is recommended as one of the most classic and effective methods because of its early appearance and deep research. The basic principle of the microinjection method is to inject the foreign gene directly into the fertilized egg (male pronucleus) with a micro-syringe, and the foreign gene is integrated into the DNA for expression using the DNA replication process in the fertilized egg split propagation. The advantage of microinjection is that the introduction of exogenous genes is more efficient and often obtains good expression. The operation is simple and easy to grasp. The injected gene is not limited by the fragment size and does not require a vector. The disadvantage of this method is that most of the exogenous genes are linked head-to-tail, and the copy number is randomly integrated into the receptor chromosomes, so the expression level is often unstable between transgenic individuals. In addition, microinjection can not introduce exogenous genes into embryonic cells at a later stage of development; microinjection is aimed at fertilized eggs, and for some single or less fetal animals, combined with low integration rates, positive offspring appear. Very few chances.