Agrobacterium-mediated acquisition of transgenic corn seeds?

1. 1 transgenic wheat 1992 obtained by gene gun method is a historic year for improving wheat by using modern biotechnology. Vasil et al. introduced GUS/Bar gene into wheat variety "Pavon" with long-term cultured embryogenic callus as explants, and obtained regenerated plants (T0) and their progeny (T 1) resistant to herbicide Basta, thus announcing the emergence of the first transgenic wheat in the world. A year later, the same research group optimized the genetic method, and succeeded in three wheat genotypes (Pavon, Bobwhite and RH7700 19) with immature embryos and their calli as explants, shortening the time to obtain transgenic wheat plants from 15 months to 7-9 months. This year, Weeks et al. also reported that transgenic wheat plants were obtained by gene bombardment with immature embryos of "Bobwhite" as explants. Two independent laboratories used the same tissue (immature embryo) of the same species (Bobwhite) and the same method (gene gun method) to obtain the same or similar results (transgenic plants), which showed that the transgenic method used was reliable. Since then, there have been more and more reports that immature embryos (or "immature embryos") and their derivatives are used as explants to obtain transgenic wheat by gene bombardment, which has become the main method of wheat gene transduction so far. It is worth mentioning that the transient expression of foreign genes and transgenic plants were also obtained by shooting the wheat embryo apical meristem, vegetative apical meristem and inflorescence meristem with "hand-held" gene gun. This method can bypass the problem of totipotent cells and has certain potential in wheat gene transduction.

1.2 obtaining transgenic wheat by pollen tube passage method; the work of transferring wheat by pollen tube passage method is mainly concentrated in the first five years (1990- 1995), and mainly concentrated in China. Zhou Wenlin and others reported that the DNA of C4 crops was introduced into spring wheat through pollen tubes to obtain transgenic wheat with some C4 traits and its offspring. Cheng Zhuomin and others claimed that transgenic wheat with CP gene of wheat yellow dwarf virus was obtained, and Zeng Junzhi and Liu Genqi also reported that transgenic wheat was obtained by pollen tube pathway method. Unfortunately, at that time, these reports lacked the molecular biological evidence of the integration and expression of foreign genes (or DNA) in transgenic wheat plants and their descendants. Recently, Zeng Junzhi and Wu et al. analyzed the expression of foreign "genes" in the progeny of transgenic wheat obtained by pollen tube pathway method during 1990- 1994, including the analysis of molecular biology methods, which proved the integration and expression of foreign genes, thus proving that transgenic wheat can be obtained by pollen tube pathway method. Zhou Wenlin, Ni et al. (personal communication) used our eMS/Bar chimeric gene to transduce wheat, and obtained herbicide-resistant and male sterile plants, and their descendants still showed herbicide resistance. Pollen tube pathway method avoids the complicated process of tissue culture and plant regeneration, and is a potential gene transduction method in wheat.

1.3 Transgenic wheat obtained by other direct methods, such as microinjection, laser microbeam puncture, PEG-mediated and electrical stimulation, is almost ineffective for wheat transformation. Although many studies have proved that foreign genes can be expressed instantaneously in protoplasts and cells treated by these direct methods, and even stably in the generated callus, there is only one report on obtaining transgenic wheat plants. How to get herbicide-resistant (PPT) green transgenic plants by electrical stimulation with protoplast of variety "Hartog" as receptor, but these plants failed to bear fruit. The difficulty in regeneration of wheat protoplasts or single-cell plants may be the main reason for the failure of direct methods such as electrical stimulation, PEG and microinjection in wheat transgenic. It is precisely because of these difficulties that the prospect of direct method with protoplast or single cell as receptor in wheat gene transduction becomes very dim.

1.4 Agrobacterium-mediated method to obtain transgenic wheat Since 1983 the first Agrobacterium-mediated transgenic plant came out, Agrobacterium-mediated method has quickly become the dominant method of gene transduction in dicotyledonous plants. The method has the advantages of simple operation, low cost, high transformation efficiency, high integration rate of single copy gene, and stable foreign gene in transgenic plant offspring. Whether this method can be introduced into gene transduction of monocotyledons, including wheat, became a hot topic of discussion and research around the 1990s. At that time, there was a tendentious view that monocotyledons were not natural reservoir of Agrobacterium, and it was almost impossible or impossible to transform monocotyledons through Agrobacterium-mediated transformation. The representative of this view is Swiss scientist Potrykus, who has made great achievements in tissue culture and gene gun transduction of cereal crops such as wheat. He once published his pessimistic views in international authoritative magazines "Biology/Technology" and "Nature". Agrobacterium-mediated method to obtain asparagus transgenic plants, especially transgenic rice and corn, not only changed the view that monocotyledons were not Agrobacterium-natural reservoir, but also proved that Agrobacterium-mediated method could be completely used for genetic transformation of cereal crops.

In wheat, although it was proved as early as 1988 that Agrobacterium can infect and carry out genetic transformation, and later it was proved that Agrobacterium can attach to wheat immature embryo cells that have been partially enzymolyzed and not enzymolyzed, transgenic plants have not been obtained for many years. Hess et al. reported that transgenic plants with antibiotic resistance and Southern blot hybridization were obtained by directly inoculating Agrobacterium into spikelets, but the existence of foreign genes was not detected in F 1 and F2. After artificial pollination, the pistil was treated with Agrobacterium (strain C 158) carrying * * * fusion vectors (pCV2260 and pSIR42). It was confirmed by PCR and Southern hybridization that pukhal'skii obtained F 1 plant, thus obtaining F2 plant with foreign gene. This simple gene transformation method combines the advantages of pollen transfer and Agrobacterium infection. If it can be confirmed by other laboratories, it will play a leading role in wheat gene transformation.

In the aspect of Agrobacterium-mediated wheat gene transduction, Cheng is equal to 1997, which breaks the silence of more than ten years and obtains normal transgenic wheat plants for the first time. Two years later, China scientist Guangmin Xia and others reported that transgenic wheat plants mediated by Agrobacterium were obtained, and the author's research group also successfully obtained transgenic wheat plants mediated by Agrobacterium (to be published). This success means that wheat can enjoy all the advantages of Agrobacterium-mediated gene transformation like dicotyledons.

"Agrobacterium-mediated method" combines Agrobacterium-mediated method and gene gun shooting method, or helps Agrobacterium to attach and transfer its plasmid into recipient cells through micropores caused by microballs, or introduces three plasmids and exogenous fragments carrying the boundary sequences of vir D 1, VirD2 and T-DNA into the recipient, and promotes exogenous purposes through the normal expression of vir D 1 and VirD2 of wheat genes in the recipient. Ultrasound-assisted Agrobacterium-mediated transformation (SAAT) helps Agrobacterium to attach and transfer its plasmid to recipient cells through micropores generated by ultrasound. Compared with the pure Agrobacterium method, the instantaneous expression intensity of foreign genes in wheat recipient cells is increased by at least 100 times. Singh, Chawla and Serik also used silicon carbide fibers to assist Agrobacterium transformation. In addition, there is an "Agrobacterium infection" method that combines Agrobacterium with virus. Although these methods are not mature at present, they have great potential in wheat gene transduction.

2. Wheat gene transformation receptor

The receptor of gene transformation depends on the transgenic method used, and the difficulty of receptor operation determines the success or failure of transgenic method.

Wheat embryogenic suspension cells and their isolated protoplasts are often used as recipients of direct transgenic methods, such as electrical stimulation, PEG and microinjection. It is precisely because of the difficulty of plant regeneration from protoplasts and suspension cells that direct methods such as electrical stimulation are not very useful in wheat gene transformation. Wheat embryogenic callus, especially immature embryo and its callus, immature embryo shield and its callus, has strong plant regeneration ability (some people think it has a large proportion of totipotent cells), which can be used as the receptor for gene bombardment or Agrobacterium-mediated regeneration of transgenic plants, thus becoming the main receptor for wheat gene transformation so far. At the same time, gene bombardment has become the main method of wheat transgenic at present, and Agrobacterium-mediated method may also become the main method of wheat gene transformation in the future. The experimental results of the author's research group in recent years (to be published) show that the regeneration ability of wheat anther callus is very strong, and it is easier to obtain transgenic plants after gene gun bombardment, so it is also a good recipient of wheat transgene. Wheat meristem, as the receptor of "hand-held" gene gun, has great utilization potential, but the development of this potential needs to be improved and is economically feasible. Pistil, as the only receptor of pollen tube pathway method and Agrobacterium-pollen pathway method, will play an increasingly important role in wheat transgenic with the improvement of these transgenic methods, and may play a leading role. Microspores, pollen, megaspores and leaf bases may further develop into recipients of wheat transgene, but they are unlikely to become the main recipients in the near future. Young spike may be a good receptor for wheat gene transformation. Chen observed that the transformation effect of young panicle was better than that of young embryo in the transformation experiment with young panicle and young embryo as receptors.

3. Target gene and selection of wheat gene transformation.

Marker genes Up to now, the commonly used selection genes and reporter genes in dicotyledonous plant genetic transformation are still the target genes and selection genes in wheat genetic transformation, which mainly include GUS, CAT and LUC. Antibiotic resistance NptII and Hpt and herbicide resistance Bar, EPSPs, Bxn, CP4 and GOX. These genes are mainly used to establish an effective genetic transformation system in wheat. With the establishment and perfection of wheat genetic transformation system, the application of true target genes aimed at improving wheat characters is increasing. Up to now, the target genes that have been introduced into wheat and expressed in transgenic plants mainly include: ① genes that improve the processing quality of wheat: high molecular weight glutenin subunit genes lAxl, lDx5, lDxl0 and recombinant high molecular weight glutenin subunit genes. ② Disease-resistant genes: coat protein gene (CP) of rice, geminoid protein (GLPs), sweet protein gene TLP, stilbene synthase gene of barley seed, ribosome-ribosome inactivating protein (RIP) gene and ds transposon of maize. ③ Chimeric male sterile genes: ribonuclease gene Barnase, cytoskeleton protein gene, etc. ④ Herbicide-resistant genes: Bar, EPSPs, Bxn, CP4 and GOX.

Antibiotic resistance genes, such as NptII and Hpt, are widely used as selection genes in dicotyledonous plants, but rarely used in wheat transgenic. There are two main reasons: one is that wheat has high natural resistance to Kan, and the other is that people are worried about the safety of antibiotic genes in wheat. So far, herbicide-resistant gene Bar is the most widely used selection gene in transgenic wheat.

The reporter genes GUS, CAT and LUC have made great contributions to the establishment of wheat gene transformation system. But today, when the wheat transgenic system is basically mature, people have begun to look for other genes to replace the above simple reporter genes or not to use the reporter genes at all. What is worth mentioning is the visual mark. McCormac et al. and Mentewab et al. used anthocyanin biosynthesis stimulating genes such as Cl/Lc as reporter genes, and selected transformants by transforming the colors of receptors and their cells. This is a reporter gene with wide application value. In addition, the synthesized green fluorescent protein gene (SGFP-S65T) has also been used in the report of wheat gene transformation.

4. Expression and analysis of foreign genes in transgenic wheat.

The promoter CaMV 35s, which is widely used to constitutively express foreign genes in transgenic plants, has a weak role in cereals. In order to enhance the expression of foreign genes in transgenic wheat, people either use double CaMV35s sequence, or add the inserter of monocotyledonous gene (such as the inserter of maize Adhl gene), or use the promoter of monocotyledonous gene, such as the promoter of rice Actl gene and the promoter of maize Ubil gene. Actl and Ubil promoters are the most commonly used constitutive promoters in wheat transgenic. In addition, the expression intensity of foreign genes initiated by artificial recombinant constitutive promoter pEmuGN in transgenic wheat is at least 10 times higher than that of CaMV35s. This recombinant promoter has great potential in wheat genetic transformation.

The application of tissue and/or organ-specific expression promoters is a great progress in wheat transgenic. Especially worth mentioning is the utilization of anther pollen specific promoters. De Block et al. used the specific promoter ca of rice anther tapetum to drive the expression of ribonuclease gene Barnse in transgenic wheat, thus creating the world's first genetically engineered male sterile wheat. The author's research group also created genetically engineered male sterile wheat by using tobacco anther tapetum specific promoter TA29. The perfection and matching of genetically engineered male sterile wheat will completely change the difficult situation of hybrid wheat seed production at present. In addition, chemically inducible promoters also have great application potential in transgenic wheat.

In recent years, there have been some studies on the integration and expression of foreign genes in transgenic wheat, including molecular research. It is generally believed that the integration mode of foreign genes in transgenic wheat is related to the transgenic methods used, but whether it is gene gun or Agrobacterium-mediated method, low copy and simple integration are still the dominant integration modes; The inheritance of introduced genes is Mendelian inheritance in most offspring of transgenic strain method. The experiment of Bieri et al. proved that the target gene (RIP) with MARs was still very stable in the last four generations of transgenic wheat, but Alvarez et al. observed that a few plants lost their integrated foreign genes in T2 generation. According to Uze et al.' s research, foreign genes can be integrated into wheat genome in the form of single or double strands, linear or circular, but the transformation efficiency of linear genes is higher. Zupan et al. observed that VirE2 protein of Agrobacterium can coordinate the uptake of single-stranded DNA in transgenic plant cells. Srivastava et al reported the method of "site-specific recombination", by which they successfully transformed four 4-copy inserted gene sites into single-copy genes.

In addition, Pederson et al also used fluorescence in situ hybridization (FlSH) to study the location of the gene introduced by particle bombardment on the chromosome of transgenic wheat. They observed that different transgenic strains of the same variety (Florida) have different insertion sites of foreign genes, for example, the insertion point of one strain is on chromosome 6B, while the insertion point of another strain is on chromosome 2A. In-depth study of this kind will help to understand the "position effect" of inserted genes, so as to better understand the expression and stability of foreign genes in transgenic wheat.

5. Main problems in wheat gene transformation.

Although the research on transgenic wheat has made rapid progress in recent years, there is still a big gap compared with dicotyledonous plants and even rice and corn, which are both cereals. The most obvious gap is that 120 varieties of transgenic dicotyledonous crops have been used in production practice or have been approved for field experiments, and several varieties of transgenic rice and corn have also been used in production practice, while transgenic wheat is still in the stage of establishing and optimizing transgenic system. The author believes that the main reason for this difference is the following problems in wheat transgenic:

First and foremost, the technical problem of wheat tissue culture. The problems of low plant regeneration rate and strong genotype dependence in wheat tissue culture are the bottlenecks that limit the success of transgenic and greatly increase the number of transgenic wheat. Wheat varieties with strong plant regeneration ability, such as Bobwhite, obtained transgenic plants by gene gun or Agrobacterium tumefaciens, while those with poor plant regeneration ability could not obtain transgenic plants by gene gun. It is precisely because of the problem of variety regeneration that transgenic wheat all over the world is concentrated on a few genotypes, which are not needed in production practice or have limited application in production practice.

Second, the receptor of gene transformation is relatively single. Immature embryos and their derivatives are the main recipients of wheat transgenic, but in developing countries around the world, few people can provide immature embryos all year round.

Third, over-reliance on otter gene marksmanship. At present, more than 90% of transgenic wheat is obtained by particle bombardment, and the shortcomings of transgenic plants by particle bombardment are already obvious in dicotyledonous plants, so I won't say much here. In fact, these two reasons are essentially caused by the first reason. In addition, the high price of gene gun is also an incidental factor.

Fourthly, there is a lack of systematic and in-depth research on the transformation mechanism of monocotyledonous plants, especially wheat, by Agrobacterium. At present, people have realized that different kinds of Agrobacterium have different infectivity to the same monocotyledonous plant, and even the infectivity of the same strain under different culture conditions is obviously different. Some monocotyledonous plants, including wheat, also have some unfavorable factors for Agrobacterium infection, such as high esterification of pectin polysaccharide in cell wall and lack of attachment sites of Agrobacterium; The secreted callus inducer is little or lacking or obviously different from dicotyledonous plants, which is not conducive to the transformation of Agrobacterium. Based on these understandings, people have made great breakthroughs in the transformation of wheat by Agrobacterium by selecting suitable Agrobacterium strains and plant expression vectors, increasing Agrobacterium concentration and prolonging infection time, and adding vir gene activators (such as As, OH-AS, dicotyledonous hemorrhage, etc.). ) culture * *, and select the appropriate recipient genotype, explants and culture medium.

Fifthly, wheat itself is an allohexaploid with a large and complex genome, and the introduced foreign genes are more silenced and modified.

Sixth, over-reliance on the role of molecular biological evidence of genetically modified wheat. In a sense, this inhibits the application of pollen tube pathway method in wheat transgenic.

6. The prospect of transgenic wheat.

With the establishment or preliminary establishment of multiple gene transformation systems in wheat, the progress of wheat transgenic research will be faster in the next five years. But this progress will be based on the establishment and improvement of a more efficient plant regeneration system. The transgenic research with gene gun as the main body will turn to the transgenic research with Agrobacterium-mediated method as the main body, which will optimize or improve the Agrobacterium-mediated wheat transgenic system. At the same time, transgenic wheat will enter the application stage from the research stage, so a small number of transgenic wheat lines or varieties will enter the field test or be released as lines and varieties. Pollen tube pathway method, especially its combination with Agrobacterium, will be widely accepted and applied in wheat transgenic practice. All kinds of auxiliary Agrobacterium-mediated methods will mature. Antibiotic resistance genes, as selection genes, will basically disappear in wheat transgenic, and will be replaced by different herbicide resistance genes, different sugar and hormone genes. The obvious and beneficial reporter genes, such as anthocyanin genes, will be widely used, so the selection efficiency of transformants will be greatly improved. More excellent agricultural traits genes will be introduced and expressed, especially those that improve the nutritional value of flour, the quality of flour processing traits, plant disease resistance, plant drought resistance and effective nitrogen utilization. Higher-intensity promoters and tissue-organ specific promoters will be widely used, chemically controllable promoters will also be used in some advanced laboratories, and single-copy insertion technologies such as "site-specific recombination" will be more perfect and applied. Genetic engineering male sterility system will be improved and used for hybrid seed production. Herbicide-resistant genes will also be used to control the purity of hybrids. In addition, there will be a clear understanding of the insertion site and insertion mode of foreign genes, so as to have a clearer understanding of the expression and silence of foreign genes. At the same time, antisense gene technology will be used to study wheat functional genes.

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I. Genetic transformation of plants

In the late 1970s and early 1980s, tobacco and potato cells were transformed with wild-type Ri and Ti plasmids to obtain regenerated plants, and then the plant genetic transformation technology based on Ti plasmid was established. In recent years, plant genetic transformation technology has developed rapidly and many transformation systems have been established. According to the methods of introducing genes into recipient plant cells, plant genetic transformation techniques can be roughly divided into two categories: vector-mediated gene transfer and direct gene or DNA transfer. The so-called vector-mediated gene transfer is the technology of connecting the target gene to a vector DNA, and then transferring the foreign gene into plant cells through host infection and other ways. Direct DNA transfer refers to the technology of transferring foreign genes into plant cells through physical, chemical and biological methods by using the biological characteristics of plant cells.

(1) Agrobacterium-mediated vector transformation is the most important vector transformation method. The modified Agrobacterium Ti plasmid can be used as a vector to effectively transfer foreign genes. There are two basic methods to obtain transformed plants: one is 1979 Marton's * * * culture method. Take plant protoplasm as receptor; One is the co-culture of leaves and discs established by Horsch et al. in 1985.

* * * culture method is a method of co-culture Agrobacterium with plant protoplasts to realize transformation. Its procedures include transforming protoplasts of primary cell walls with Agrobacterium, screening transformed cells, inducing differentiation of transformed cells and plant regeneration.

Leaf disk method is actually a transformation method created by improving the culture method. Infection of leaf explants with Agrobacterium and short-term culture. In the process of culture, the vir gene of Agrobacterium is induced, and its activation can start the transfer of T-DNA to plant cells. After * * * culture, it is necessary to screen and transform explants, culture callus, induce differentiation and other steps to obtain regenerated plants. Leaf disk method is a good method to use plant explants as transgenic materials, because it does not need protoplast operation and it is simple and fast to obtain transformed plants.

Agrobacterium-mediated genetic transformation is a common method to transform most dicotyledonous plants. However, monocotyledons are rarely infected due to the host limitation of Agrobacterium, especially some important crops such as rice, wheat and corn are not sensitive to Agrobacterium, so it is difficult to apply this method for genetic transformation.

In addition to the above-mentioned gene transformation with Ti plasmid as the vector, liposomes can also be used as the vector for gene transformation. Liposomes are membrane-like structures composed of phospholipids, so DNA molecules can be wrapped in liposomes to avoid the degradation of dnase in recipient cells and introduced into plant protoplasts.

(II) Methods of direct gene transfer This refers to some gene transfer methods that do not depend on Agrobacterium or other vectors or media.

1. Electrical stimulation and injection can change the permeability of cell membrane. The method of introducing exogenous DNA into plant protoplasts by high voltage electrical pulse electrical stimulation perforation is called electroporation. This method has been widely used in gene transfer of monocotyledonous and dicotyledonous plants and animals. For example, in the late 1980s, neomycin phosphotransferase (NPT) gene was transferred into protoplasts of maize inbred lines by this method, and plants were regenerated.

The method of directly introducing genes into intact plant cells or tissues by electrical stimulation technology is called electrical injection. This method avoids the complicated operation and difficulties of protoplast culture and plant regeneration, and has great application potential.

2. Gene marksmanship Gene marksmanship is also called particle bombardment. This method uses a particle gun to shoot metal particles with exogenous DNA adsorbed on their surfaces into plant cells or tissues at high speed. Because this method is fast and simple, not limited by the host range, and the recipient plant cells do not need to remove the cell wall, the transformation rate is high, and the transformed cells or tissues are easy to regenerate plants, so it has attracted much attention.

3. microinjection microinjection is the direct injection of foreign genes or DNA into the nucleus or cytoplasm by mechanical means using a microinjection instrument. Compared with other methods, this method is more direct and effective in introducing foreign genetic material.

Microinjection is not only used for plant cells, but also developed to directly inject plant ovaries in recent years, which is more conducive to the transformation of exogenous genetic material into young embryos. Since 1970s, China has explored this field in theory and practice, and obtained cotton transformed plants with resistance to Fusarium wilt and insect pests.

Second, transgenic animal technology and its application

(1) The concept of transgenic animals Through genetic engineering technology, foreign target genes are introduced into germ cells, embryonic stem cells and early embryos, and are stably integrated on the recipient chromosomes, so that individuals who can pass on foreign target genes to future generations can be obtained through various development channels, that is, transgenic animals. The transferred target gene is called transgene, and this process of transferring target gene is called transgene. The concept of "transgenic" is usually limited to gene transfer in animals and plants through genetic engineering technology, so the way of obtaining new genes through sexual or asexual hybridization between varieties does not belong to this category.

(II) Transgenic animal technology Transgenic animal technology is a biotechnology developed in the early 1980s, which overcame the reproductive isolation among species and realized the exchange and recombination of genetic materials among animal species. According to the different methods and objects of exogenous gene introduction, there are three main ways to produce transgenic animals, namely microinjection, retrovirus and embryonic stem cells. The basic methods of retrovirus gene transfer have been briefly introduced, and only two methods are introduced here, microinjection and embryonic stem cell method.

1. Prokaryotic microinjection of fertilized eggs is developed from the nuclear transfer experiment of animal embryology. In the early 1980s, people used this method to transfer foreign genes into animal germ cells and successfully established transgenic mice. Transgenic sheep and pigs are 1985. Since then, transgenic mice, rabbits, chickens, cows, fish and so on. Success again and again. It can be seen that microinjection is a widely used and most effective technology to obtain transgenic animals.

Under the microscope, a glass tube with a diameter of about 1μm was directly inserted into the male pronucleus of the fertilized egg, and the exogenous gene DNA in the capillary was injected into the pronucleus, and then transplanted into the fallopian tube or uterus of the pseudopregnant mother to develop into offspring. In order to understand the integration of transgenes, dot blot hybridization, polymerase chain reaction or Southern hybridization can be used to detect the DNA of offspring individuals.

The advantage of microinjection method is that the introduced foreign gene fragment can be as long as 50 kb without vector, so the integration rate of foreign gene on host chromosome is relatively high. Its disadvantage is that the integration of foreign genes is random, so it is difficult to control its integration rate; Moreover, whether the foreign gene can be stably integrated into the recipient genome can only be determined after the offspring are born, which is not conducive to the application in large domestic animals with long growth period and small litter size.

2. embryonic stem cell (ES) refer to undifferentiated embryonic cells separated from the cell mass in the blastocyst stage of mammalian embryos, which have developmental totipotency and can differentiate into various tissues. In the mid-1980s, people began to study the method of obtaining transgenic animals by using ES cells. In this method, exogenous genes are directly introduced into es cells, screened in vitro, injected into the blastocyst cavity of the recipient, aggregated with the blastocyst cells, and become a part of the recipient embryo to participate in its differentiation. Transgenic animals with chimeras developed from this embryo, that is, some tissues came from donor es cells that integrated foreign genes. In the process of chimerism, germ cells differentiated from transformed ES cells can pass on the introduced foreign genes through hybridization.

In the technology of obtaining transgenic animals from embryonic stem cells, foreign genes can be introduced into es cells in many ways, and the identification and screening of cells are convenient. The copy number, location, expression level and insertion stability of foreign genes can be determined in advance at the cell level, and the operation of injecting ES cells into blastocysts is simple and the integration rate is relatively high. It's just that the establishment of ES cell line itself is a very difficult task. Although mouse ES cell line has been established, it has not been able to obtain a truly stable ES cell line in pigs and sheep.

(III) Application of Genetically Modified Animals The research content of genetically modified animals is very extensive. Since 1980s and 1990s, there has been great development from basic theory to applied technology, and it has gradually moved from laboratory to production practice.

1. Application in the study of gene expression regulation Transgenic animals can be used as "reactors" to study the regulation of foreign gene expression in vivo. Here, the cis-regulatory elements of DNA and the spatio-temporal regulation of developing genes are introduced as examples.

Studies on (1) cis-regulatory elements Abnormal lipoprotein levels are related to diseases such as atherosclerosis. Humans have five kinds of lipoproteins, each of which contains a different apolipoprotein. At present, about 17 kinds of apolipoprotein have been found and their coding genes have been sequenced, which are candidate genes for studying cardiovascular diseases. Transferring apolipoprotein gene into mice can be used to study human lipoprotein metabolism, regulation and atherosclerosis. When studying the cis-regulatory elements of tissue-specific expression of apolipoprotein genes, two clusters of tissue-specific expression of apolipoprotein genes were found (figure 19- 15). A cluster including genes A-Ⅰ, C-Ⅲ and A-Ⅳ is located in the second and third regions of human chromosome111q 23, and consists of a-Ⅰ, c-Ⅲ and a-Ⅳ. The transcription direction of C-ⅲ is opposite to that of the other two genes. A-ⅰ and C-ⅲ are mainly expressed in liver and small intestine, and A-ⅳ is mainly expressed in small intestine. The range of C-Ⅲ gene -0.2 ~- 1.4 BP is the region that regulates the expression of A-Ⅰ gene in small intestine. This region is also a regulatory element for the expression of C-Ⅲ and A-Ⅳ genes in small intestine, indicating that this element can regulate the expression of the whole apolipoprotein gene cluster in small intestine. The other gene cluster is located in the long arm 3 region of chromosome 19 (19q 13), and contains E, C-I and C-II genes, arranged in the order of E, C-I and C-II. E gene is mainly expressed in liver and most tissues, but the expression level is low. Later, it was found that there was a gap between C-ⅰ gene and C-ⅱ gene.