Transgenic animal

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Genetically modified animals refer to animals cultivated by genetically modified engineering.

The basic substance of heredity is DNA Genes are DNA fragments with genetic effects located on chromosomes chromosome All in genetic information , which can be called genome. Since the biological gene composition of different species and individuals is different animal For individuals, non self genetic components belong to foreign gene If foreign genes are integrated or introduced into animal chromosome genes, then this foreign gene Is called transgene (transgene) Transgenic animal （transgenic animals）。

The expression system of transgenic animals includes foreign genes, expression vectors and receptor cells. The transfer of genome is Nuclear transfer And animal cloning technology, artificial synthesis and design of genes, whole genes and even genomes transgenic technology yes Synthetic Biology 。

Chinese name: Transgenic animal
Foreign name: transgenic animals

Meaning: Integration of recombinant gene transfection into animal receptors
DNA: inheritance Basic substance of
Genes: There are inheritance DNA fragment of the effect

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Golden Egg Project of Genetically Modified Poultry

Mammals The gene transfer method is to use the reconstructed target gene (or genome segment) Microinjection Such methods as injecting the fertilized egg (or pre implantation embryonic cell) of the experimental animal, and then implanting the fertilized egg (or pre implantation embryonic cell) into the recipient animal's Fallopian tube (or uterus) so that it can carry foreign gene Of transgene animal.^[1]

According to the different methods and objects of foreign gene introduction, the main methods of producing transgenic animals are Microinjection 、 Retrovirus Law embryonic stem cell (Embryonic stem cell, ES cell) method Electric pulse Method, sperm carrier introduction method, etc.

Microinjection

Is the most commonly used method with high success rate transgene mice As an example, the general process is as follows.

1、 Collect fertilized eggs

（1） Superovulation : Select 6~8 Weeks old Of female mice, successively intraperitoneal injection 5U Pregnant Horse Serum Gonadotropin (pregnant mare serum gonactotropin, PMSG) and 2.5~5.0U Human chorionic gonadotropin (humar chorionic gonactotropin, HCG) to promote ovulation, and then mate with male mice aged 12 weeks to 1 year at 1:1. The sign of successful mating is in the female mice A white vaginal plug was found at the vaginal orifice.^[2]

(2) Fetching fertilized eggs: the next morning after the cage is closed with male mice, the female mice confirmed to have vaginal plugs are killed by cervical dislocation method, and taken out Fallopian tube , placed in Culture medium In, in Stereo microscope Carefully cut the ampulla of the swollen fallopian tube with Hyaluronidase Flush the solution with a little force to separate the fertilized egg from the fallopian tube, and flow it into the culture medium. Select it under the stereomicroscope Prokaryote Clear fertilized eggs, transferred to embryos with culture medium Petri dish Then drop mineral oil into the culture dish to cover the surface of the culture solution, and put it in the CO2 incubator (37 ℃, 5% CO two , 95% air) for about 5 hours (generally, the fertilized eggs are taken at about 10:00 am, and the female and male pronuclei can be seen at l3, and most fertilized eggs are usually formed and clearly visible between 13:00 and 18:00).

2、 Inject DNA solution into the male pronucleus of the fertilized egg

In the fertilized egg, there are two pronuclei from the egg and sperm respectively. Generally, the male pronuclei from the sperm are larger and can accommodate more foreign DNA, so DNA solution is usually injected into the male pronuclei; In addition, the gene DNA to be imported must first be used agarose gel electrophoresis Determine its purity. Will contain 10-20 fertilized eggs Culture medium Drop and DNA drop are dropped together at Slide And then fixed on Microinjection On the stage of the instrument, set the center of the field of vision on the DNA liquid drop, hold a glass straw filled with mineral oil in the right hand to absorb the DNA solution. The amount of inhalation shall be subject to the crescent shaped position between the DNA solution and mineral oil, and then move the center of the field of vision to the fertilized egg liquid drop. The left hand controls the holding tube, and uses negative pressure to fix the fertilized egg, And move the tip of the straw on the right hand to the fixed fertilized egg, and then insert the fertilized egg male Prokaryote The DNA solution is injected into the male pronucleus. To confirm whether it has been injected, it is necessary to determine whether the male pronucleus has expanded with naked eyes. Only undamaged eggs that have completed the operation are collected and incubated overnight in a 37 ℃ CO2 incubator. On the second day, eggs that have developed into two cells are selected.

3、 Transfer the 2-cell fertilized egg to the false pregnant female mice Of Fallopian tube in

Male mice with vasectomy and female mice in estrus (generally ICR mice ）Although mating can not cause fertilization, it can stimulate the cervical canal, activate the corpus luteum in female mice, and create an endocrine environment that can continue pregnancy. This kind of mice is called pseudopregnant female mice. Transfer the microinjected fertilized eggs to the donor petty The oviduct of the 3 day pseudopregnant female rats can still develop normally. During operation, it is better to transplant both fallopian tubes at the same time. In this way, when the condition is good, 8 offspring can be obtained, and sometimes only 1 offspring can be produced. The damage caused by DNA injection can make many fertilized eggs stop developing halfway.

4、 Identification of introduced genes

Generally, embryo transfer About 20%~30% of all offspring born have introduced genes, so Southern Blot or PCR method should be used to genetic genes Conduct analysis to select and produce external sources Sex gene Successfully imported mice Breeding and reproduction.

gene Microinjection Is characterized by foreign gene The import and integration efficiency of is high, no carrier is needed, and the transfer is direct Target gene The length of the target gene can reach lOOkb (100000 base pairs). It can be directly obtained Pure line , so the experiment period is short. However, expensive and precise instruments are needed, technical operation is difficult, and integration sites and copy number Are uncontrollable and easy to cause Insertional mutation , causing corresponding character If you change, you will die.

Retrovirus Infection method

Retroviruses are invasive host cell And integrated into cells Chromosome DNA Ability. take foreign gene DNA insertion Retroviral vector , packaged into highly infectious virus particles through auxiliary cells, after infecting the embryo, the infected morula embryo cells are introduced into the uterus, and can develop into a virus carrying foreign genes Progeny animal.

This method has a high integration rate gene It is not easy to destroy, mostly single copy, unit point consolidation, suitable for difficult to observe Prokaryote Of birds. Because of the virus capsid The target gene should not exceed 10 kb due to the size restriction, otherwise the activity and stability will be affected. In addition, viral DNA may affect the expression of foreign genes in host animals.

Embryonic stem cell method

embryonic stem cell (ES cells) are derived from Blastocyst stage Of Inner cell mass The undifferentiated embryonic cells isolated from Omnipotence , can be cultured in vitro<; Amplification, transformation and genetic production Mutant etc. Genetic manipulation 。 This law integrates foreign gene ES cells as donor somatic cell The general process is as follows:

(1) Embryos that have developed to a certain stage are obtained. After culture, inner cell clusters are stripped and dispersed, and then re cultured. Finally, ES cells are separated, diffused, and identified.

(2) Via Gene targeting technology , the exogenous gene is retrovirus infection 、 Electric pulse Methods, etc, in vitro culture And screening gene expression The.

(3) Embryos at blastocyst stage were obtained as recipients of ES cells.

(4) ES cells were injected into Blastocyst stage The cavity of the embryo, which is closely connected with the inner cell mass, becomes Chimera 。

(5) After the injected embryos are cultured, the blastocysts without developmental defects are screened and transferred to the petty In the uterus of 3-day pseudopregnancy receptor animals transgene animal. This law foreign gene The integration rate is high, and suitable transformed ES cells are screened before implantation into blastocysts, which overcomes the problem of only Progeny It is a promising technology to make full use of various advanced methods developed in molecular biology. The disadvantage is that it is not easy to establish ES Cell line 。 And because of the chimeric approach, the experiment cycle is long.

Electric pulse method

Electric pulse Electroporation, also known as electroporation, is the process of combining donor DNA with Receptor cell Fully mix, change the cell membrane structure under the external high voltage short pulse, make the cell membrane produce instantaneous reversible electroporation, so that a certain size of DNA can enter the cell through the cell membrane and be transported to the nucleus. In 1980, Zimmermann and others first used electric pulse technology to guide drugs and dyes mice Thymocyte In the same year, T.K. Wang and Neumail first reported that TK gene was introduced into CTK cells of CTK mice by electric pulse method, and 67 transformed clones were obtained from 106 treated cells: foreign gene Electric pulse method. In animals, electric pulse method is mainly used to transform embryonic stem cell 。^[3]

Sperm introduction

utilize sperm As an external source Gene vector , with Fertilization The method of introducing foreign genes into the fertilized egg and integrating them into the genome of the fertilized egg is called sperm vector introduction transgene A new attempt by animals. This method is simple and convenient, relying on the physiological banding process, eliminating the need for Prokaryote Damage. However, in practice, the success rate is low, and there is controversy about whether sperm can be used as a foreign DNA carrier. This technology is still in the exploratory stage. This method can combine artificial insemination, in vitro fertilization and transgene.

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Basic theory

Developmental biology

transgene Animals can be used to observe different target genes in embryos Developmental stage Of Specificity Expression, shutdown and regulation mechanism, understanding the regulation sequence (such as Enhancer 、 Promoter ）On Tissue specificity The role of the human renin gene in mice The specific expression in vivo may be related to the 5 'flanking sequence of the gene. In addition, transgenic animals can also be used to identify genes (including Endogenous gene ）It can also measure the expression characteristics of unknown genes related to animal development.

genetics

utilize Natural mutation Or artificially modified genes as foreign gene , construct transgenic animals and study the phenotypic effects of abnormal genes in humans, which can understand the relationship between gene structure and function, and can also be used to Genomic imprinting Analysis Genetic defect Correction, etc.

Medical research

Cardiovascular disease

Various factors regulating cardiovascular function, such as trans lipoproteins, trans Fibrinolytic enzyme The original class can pass transgene Animals to understand their physiological functions and functions, and establish atherosclerosis , sudden hypertension, venous occlusion and other transgenic animal models.

oncology

The discovery of tumor genes is a major breakthrough in oncology research in the past 10 years. More than 100 tumor genes, including breast cancer genes, have been found. Experiments have proved that all kinds of vertebrates carry tumor genes, which usually do not cause cell canceration Only under certain conditions can it be activated to make cancer cells proliferate and cause canceration. The establishment of transgenic animals with tumor genes can understand which tissues have an effect on tumors Gene transformation Activity sensitivity, the relationship between tumor formation and its genes, the influence of tumor gene growth and differentiation, etc.

Hereditary disease

It is usually the external source that will function normally Gene introduction In target cells of animal body, genes used to make up for defects and change diseased cells genetic material , gene therapy. On the contrary, the dominant disease gene or one or more foreign gene The genetic disease can be prepared by artificial introduction into animals transgene Animal models, research and treatment Human genetic diseases 。 For example, Huntington introduced chorea genes mice , established an animal model of chorea, and Redhead incorporated the MBP of normal mice（ Myelin The tremor symptoms of mice disappeared when basic protein (ALP) gene was introduced into tremor mice.

immunology

Babinet found that although transgene The mice produced HBsAg, but there was no pathological change within 6 months, showing a continuous viral status. These results indicate that: hepatitis B Patient's Hepatocyte injury It is not directly caused by the HBsAg expression of HBV, but by the immune reaction to the viral antigen on the liver cell membrane. Therefore, you can use transgenic mice Model to study the relationship between immune tolerance and hepatocyte injury to explore the pathogenesis. In addition, transgenic mice are also classified as Class I and Class II Major histocompatibility antigen The function of provides new means.

Improved cultivation

Classical genetic breeding methods can only be carried out among species of the same species or closely related parents, and are limited by variation or mutation Recombinant DNA technology Interspecies that can make the genetic relationship far away in a short time genetic information Exchange and reorganization. In addition, due to transgene Animals can integrate stably foreign gene , and expressed in appropriate tissues character It can be passed on to future generations, so that transgenic livestock can be produced with faster growth, more meat, wool and milk production and less material consumption, providing an important way for livestock improvement.

Development and production

Will be valuable in the medical field biological activity The fertilized egg of a domestic animal or poultry, which is transformed into transgene Harvested in animal body fluid or blood, milk, urine and ascites Gene product A large number of valuable bioactive proteins can be obtained. This animal is usually called“ Animal bioreactor ”。 Among them, milk is the most ideal secretory site. TPA (organizational type Fibrinolysis Protein element Activation factor ）Transgenic mice It has been expressed in the milk of rats and has become an ideal drug for the treatment of thrombosis. Beta milk Globulin In transgenic mice, β - lactoglobulin, the main component of goat milk, can be expressed. In addition, coagulation factor IV and α 1- Antitrypsin It was also expressed in transgenic sheep. Others such as hepatitis B virus Antigens Follicle stimulating hormone 、 Luteinizing hormone They can also use transgenic animals to produce as needed, opening up a very broad world for the development of medicine, food and animal husbandry.

Disease model

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Animal models of diseases have contributed to the development of medicine. However, many diseases are difficult to create animal models by artificially induced methods, or many diseases do not occur in experimental animals or are only high Mammals Only occurs, so it is difficult to automatically or manually Directional cultivation The animal model was obtained by the method of. transgene The emergence of technology has helped human beings accurately study genes and diseases Correlation It is possible to Ontogenesis Any individual is used for analysis of genetic function in each stage of. Therefore, the development of transgenic disease animal models has become a hotspot of transgenic animals, some of which have entered the application stage.

Virus model

（1） Polio virus recipient transgene mice To clone human poliovirus receptor and make transgenic mice. Human PVR gene of poliomyelitis virus Microinjection In the early embryos of C57BL/10 mice, transgenic mice were produced and finished lines were bred. This kind of mouse expresses human receptor, which has poliovirus Receptivity 。 In addition, the mice infected with this virus showed the same clinical symptoms as human beings Specificity They also show the same nature as people. Therefore, this kind of mouse is not only a human disease model, but also may replace monkey It is widely used to test the efficacy and specificity of poliovirus.

（2） Hepatitis B Virus carrier model: The incidence of liver cancer in hepatitis B virus (HBV) carriers is 100~200 times higher than that in normal people, but there is still no effective treatment. HBV only infects humans or gorillas, and no other suitable animal models have been developed. It is generally believed that the immune response to HBV is dominated by genes, and those with insufficient immune response become chronic hepatitis ； The mechanism of liver cancer is not single. There are various problems of necrosis and regeneration of liver cells caused by chronic hepatitis genetic variation And canceration occurs. HBV genome is a ring containing some single stranded regions Double chain DNA molecule , the length of two single chains is different, the long chain is negative (3.2kb), and the short chain is Positive chain About 50%~80% of the negative chain. Therefore, if the l.2HB-BS DNA is made into linear DNA with two ends repeated for transduction, the whole genome can be expressed. On the other hand, when only HBS antigen expression is required, only 1.2 HB-BS genes need to be introduced. The HBV DNA added with 1.2HB-BS was introduced into C57BL/6J mice , HBV replication in liver, release in blood Virus particle 。 Gene expression occurs in the embryonic stage, but it shows immune tolerance (passive state) to these viral antigens, without any pathological changes, so it can be used as a model for human HBV carriers. Human transgenic mice, like humans, have no clinical abnormalities.

（3） hepatitis B surface antigen transgene Animal model: human hepatitis B surface antigen（ HBsAg ）Transgenic mice with HBsAg gene can be obtained by gene introduction into mice transgenic mice HBsAg can be produced in the liver of. The transgenic mice can not only mimic the Virulent state It does not cause disease. Chisari found that the HBsAg positive transgenic mice immunized with HBsAg plus complete or incomplete Freund's adjuvant could not induce specific antibodies, while the HBsAg negative transgenic mice responded. The HBsAg positive transgenic mice did not show any pathological changes within 6 months, but showed a persistent virus carrying state. The results of these tests show that patients with hepatitis B Hepatocyte injury It is not directly caused by the expression of HBsAg, but by the immune response to the viral antigen on the liver cell membrane. This transgene mice The model can be used to study the relationship between immune response tolerance and hepatocyte injury, and explore the pathogenesis and persistence Virulent state There are many problems related to HBV pathology and therapeutics, such as clearance, drug screening experiment, replication, expression and regulation of HBV DNA in the host and the relationship with the pathogenesis of hepatitis B.

In addition to the above transgene In addition to the establishment of mouse animal models, there are some other viral diseases transgenic mice Animal models have also been established. For example, JC injection Viral genome Obtained transgenes mice , which can be used as a transgenic mouse model of progressive multiple leukoencephalopathy (PML) T lymphocytes The transgenic mice prepared by the tyrosine aminotransferase (TAT) gene of type l virus (HTLV-1) can be used as the disease animal model of human nerve fiber.

Tumor model

Mammals 's DNA carries Oncogene (oncogene), in the physical and chemical or Biological factor It is activated under the action of cell proliferation , abnormal regulation of differentiation and disorder of relationship with surrounding tissues, leading to canceration. Making tumor with oncogene or oncovirus gene transgene Animal model, which can explore the relationship between foreign oncogenes and the Proto oncogene (susceptible protooncogene), oncogene expression and cancer transformation, oncogene expression and animals genetic background Or external activation factors.

Through to mice Incubation of fertilized eggs by inserting oncogene or proto oncogene transgenic mice It can study the influence of oncogenes on normal cell division and differentiation at the overall level, so as to accurately study the relationship between oncogenes and tumor formation. For example, the SV40T antigen gene is widely studied in transgenic mice Transformant gene 。 Brinster et al Promoter After the sequence was connected, it was introduced into mice and found that foreign gene Can be used in mice central nervous system Give priority to expression and cause Choroid plexus papilloma 。 The recombinant molecules linked with T antigen sequence and different promoter sequences can also cause tumors in the tissues expressed by promoter sequence after being introduced into mice. These tissues are pancreas, liver, eye lens, and even Myocardial tissue Etc.

In addition, like Viral oncogene , Cells Proto oncogene After connecting with different promoters, it is also imported mice And obtain transgene animal. If will C-myc Oncogene Placed in mice Breast tumor Under the regulation of virus (mMTV) gene promoter transgenic mice 。 These mice had lymphoid tissue And other parts can cause tumors. These results show that abnormal regulation of proto oncogenes has a tendency to make tissues prone to deterioration. The above SV40T antigen gene and c- Myc gene The research results of mice strongly support the hypothesis of multi-step tumorigenesis, that is, the tumorigenesis requires at least secondary transformation. For example, T antigen and c-myc gene can be expressed in all organs, but only in a few organs canceration occurs.

Metabolic model

(1) Gaucher disease transgene Animal model Gaucher disease is Glucoside Enzyme defect（ Lysosomal enzyme A metabolic disease that causes the accumulation of visceral glucose cerebrosides. According to clinical symptoms, it can be divided into three types, including adult type (type 1) and subacute youth type (type III) Hepatosplenomegaly Or anemia osteoporosis The acute infantile type (type II) showed central nervous symptoms such as spasm. Gaueher cells (macrophages containing glucocerebrosides) appear in liver, spleen, lymph nodes, bone marrow, etc. of all types. Spontaneous nude mice and experimental induced models have been reported, but the research on treatment is still insufficient.

reform mice β - glucosidase gene, and carry out Gene targeting 。 stay Exon Neopenicillin was constructed between 9 and exon 10 Phosphotransferase Resistance gene (near) or construct a targeting vector with HSV tk at the 3 'end, and implement PNS in ES cells（ Positive and negative selection Method). Afterwards, the chimeric mice were bred and analyzed for homozygotes or heterozygotes. β with normal individuals- Glucosidase The activity of heterozygotes was 44%, Homozygote 4%. Homozygote showed the accumulation of p-glucocerebroside. Homozygous individuals with normal or higher size Heterozygote Significantly small, presenting dyspnea , cyanosis. Such individuals were not raised by female rats and all died within 24 hours. Pathology shows macrophages in liver, bone marrow, brain and spleen lysosome The accumulation of internal fat is the same as human pathological changes, but this mice The differentiation of blood cells can be seen in the early fetal liver, although macrophage There is fat accumulation in lysosomes, but sudden death is rare. It can be considered that it is consistent with the acute neurological symptoms seen in children with Gaucher disease type II.

The model is an existing transgene One of the highest evaluated and applied gene models in disease animal models.

（2）FAP transgenic mice

Familial polyneuropathy Amyloidosis (FAP) is Autosomal dominant inheritance It is a disease characterized by peripheral nerve and autonomic nerve damage. Amyloid protein The main component of the amino acid mutation occurred. Most patients were replaced with methionine at the 30th valine position, which has been proved to be the replacement of the base of the gene corresponding to the amino acid mutation. Therefore, in terms of etiology, it is a disease that protein variation directly leads to amyloidosis. Construction of variant amyloid gene with MT promoter of metallothionein gene transgene Methods integrated into C57BL/6J mice The analysis results of the transgenic mice showed that the mutant amyloid protein gene expression It can be seen from embryonic stage, while variant amyloid deposition occurs in adolescence, and then the amount of deposition gradually increases with age.

However, the disease occurs in the peripheral nerve or autonomic nerve Amyloid protein Calm, but in transgenic mice Does not appear in. Whether this is due to differences in histological characteristics between mice and humans is unclear. For this reason, transgenic mice do not show clinical symptoms.

From the analysis of these mice, the following four points have been clearly understood: ① The changes of the tetrameric complex composed of the transhydrin molecule in the blood are important for the deposition of amyloid; ② The tiny component of amyloid protein, namely serum amyloid protein P, does not affect the beginning, extension and tissue distribution of amyloid deposition in any way; ③ The microenvironment of each tissue, i.e. blood flow abundance and tissue density, has a greater impact on the amount of amyloid deposition; ④ The feeding environment of mice affects the sedimentation of amyloid protein. This transgene mice The key problem is that amyloid deposition does not occur in peripheral nerves and autonomic nerves. Clarification of this problem will be important for the development of treatment methods in the future.

The study of transgenic disease animal models is mostly limited to the transfer of a single gene. The performance of biological functions and the occurrence of diseases are usually the result of the interaction between genes, so the research results in diseases and TCM syndrome types Functional genomics , using transgenic technology It is more meaningful to make functional genome animal models. The development of functional genome animal models will follow human genome project And gradually realized.

gene therapy

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Gene therapy ready for use Molecular Biology Technology take foreign gene To introduce target cells to correct and compensate gene defects or inhibit and block the overexpression of abnormal genes, so as to achieve the purpose of treating diseases. Gene therapy includes gene compensation, gene correction Cytokine gene Introduction, antisense RNA technology, etc. As a new method to treat diseases, this technology has developed very fast, and several cases have entered the clinical practical stage, solving clinical problems that cannot be solved by traditional methods. This novel and unique treatment method also originated from transgene mice Research.

Animal model making of gene therapy, commonly used Retrovirus Introduction of foreign sources for carrier method Target gene 。 This recombinant retrovirus can transfer the functional Gene integration reach Receptor cell On chromosomes, the expression products of imported genes will make up for the original lack of gene products. A mouse lacking growth hormone, usually smaller and male sterile than normal mice. take growth hormone Gene transfection into this kind of mice, through the expression of exogenous growth hormone gene, can make the original "dwarf" A and mouse individuals grow three times, and can restore the male Fecundity ； lack Major histocompatibility complex (major histocompatibility complex, MHC) mice lack immune response to synthetic anti Ig, but MHC transgenic mice can restore immune response; With β Thalassemia The degree of anemia was alleviated by introducing mouse or human 8-globulin gene into 30% mice.

The production of gene therapy animal models can also use import Antisense gene method. This method is suitable for diseases caused by abnormal expression of some genes. Specifically Antisense DNA Inject the fertilized egg and integrate it into Chromosome And express RNA complementary to pathogenic mRNA sequence Double chain Make pathogenic mRNA untranslatable. Human nerve tremor is a disease caused by the decrease of myelin basic protein (MBP). Integrate antisense DNA of MBP into mice The MBP synthesis of chromosome genes was reduced to 50%~70% of the normal level, so tremor occurred, and the tremor animal model was made. MBP deficient mutants were also found in mice, showing spontaneous tremor. On the contrary, when MBP DNA is transferred into these mutants Myelin Basic protein. When the mRNA expression amount reaches more than 25% of the normal MBP amount, the symptoms disappear, showing obvious symptom recovery (treatment) and the relationship between the disease and the MBP expression level transgene Animal production provides another way.

Research progress

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The transformation of biological populations in nature by human beings begins with artificial screening and breeding, and then artificial hybridization Artificial mutagenesis 。 In 1983, Chinese scientists Zhu Zuoyan It is the first internationally developed Transgenic fish 。 In the 1990s, China successfully developed transgene Sheep.^[4]

Transgenic poultry Bioreactor , including liver and Fallopian tube Expression system. In 1993, Dr. Sang of Ruslin Institute successfully expressed foreign proteins in egg yolks. 1994-1995 China Zeng Bangzhe Put forward the method of system genetics and pioneered Oviduct bioreactor Concept, terminology, vocabulary, etc. – Golden Egg Plan for genetically modified poultry. In 1996, the 1st International Symposium on Genetically Modified Animals (Zeng Bangzhe, Secretary General) was held in Beijing, and Zeng BJ expounded on fallopian tube bioreactor, biosystematics and genetics bioengineering And got Zhu Zuoyan Sunrise drying 、 Liu Depei With the participation and support of famous Chinese scientists, mammalian mammary gland bioreactors and poultry oviduct bioreactors have become important directions for transgenic animals.

Animal cloning – Asexual reproduction The technology began in 1938 with the ligation experiment of salamander fertilized eggs by German scientist Sperman. 1961 Chinese scientists Zhu Xi Amphibians were successfully studied by artificially stimulating mature toad eggs Artificial parthenogenesis 。 In 1962, British scientist J B. Gurdon， Successfully cultivated by nuclear transplantation Xenopus Laevis Adult. In 1980, American biologist P C. Hoppe and Geneva Super microsurgery expert K I. Illmense， use blastocyst The nuclear transfer method has successfully bred mice 。

In 1997, I Wilmut et al., using cells from sheep mammary gland cells nuclear transfer To enucleated Egg cell Medium, successful cloned sheep Dolly, the main purpose of cloning animals is to solve the problem of Bioreactor Of transgene Pure breeding of animal strains.

In the 21st century, genetic engineering synthetic biology based on system biology, that is Systems Bioengineering Technological progress, transgenic technology Including the research and development of transgenic animals, including artificial design, gene synthesis and gene regulation Transgene of chain System biotechnology The new era has changed the previous single Gene cloning 、 Carrier construction And Gene transfer Method, opened Whole gene synthesis , Genes Regulatory network Design and even labor genome Transferred polygenes Transgenic organism The age of technology.

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