Chapter 4 2

According to the central dogma, the development of an organism is the expression of the genetic information contained in the DNA molecule under certain conditions.In other words, the individual development of an organism proceeds according to instructions (the genetic program) that are somehow pre-established in the DNA molecule.These instructions, under the right conditions, can cause a cell or certain cell populations to differentiate at exactly a specific time and location (space).From this point of view, the genetic information contained in DNA is the internal basis for the development of organisms, while the physiological state and various environmental factors in the body are the necessary conditions for development.As for which part of a cell's full genetic potential is expressed during development, it depends on which genes are turned on and which genes are turned off.About 50 years ago, biologists realized that during development, when cells differentiate, some genes must be turned on and others turned off, so that it is possible for cells from the same fertilized egg to proliferate, some develop into lungs, and some Some developed into hearts, while others developed limbs... Obviously, there is a problem with the regulation of gene expression here.

As early as the 1940s, when American geneticist McClintock was studying the high-frequency variation of corn kernel color, he had already noticed the problem of gene regulation, and used the transposon theory to explain the genetic instability of corn kernel color. The gene regulation model was proposed, which initially revealed the mystery of how organisms design and arrange gene activities.In addition, in the 1940s, there was the theory of one gene and one enzyme proposed by Biddle and Tatum in 1946, which clarified that genes control the development of traits through enzymes, and then drew people's attention to the relationship between genes and enzymes. .

In 1961, French biologists Jacob and Monod (J·Monod, 1910-1976), when studying the regulation mechanism of galactose metabolism in Escherichia coli, proposed the operon theory to further develop and deepen the mechanism of genes acting through enzymes. Mechanism, creating a gene regulation model at the molecular level, has opened up new ideas for revealing the development of organisms and cell differentiation. According to the operon theory, genes can be divided into several types, one is the structural gene (indicated by at), which contains information about the protein structure; the other is the regulatory gene (indicated by RG), which has the function of adjusting the activity of the structural gene, It can restrict the formation of a repressor (a small molecular protein) that suppresses the activity of a structural gene under normal circumstances; the third is the operator gene (indicated by O), which itself cannot produce anything. , the structural gene cannot be transcribed.In addition, there is a promoter gene (indicated by P), which is the place where RNA polymerase is accepted and the starting point of RNA polymerase activity. RNA polymerase starts to transcribe the structural gene from here.The so-called operon refers to the sum of a series of structural genes and operating genes that are closely related in function and arranged together.The switch of the operon is inseparable from the role of the regulatory gene and the operator.

For example, E. coli breaks down lactose into glucose and galactose.The enzymes that catalyze this biochemical reaction are galactosidic acid, lactose permease, and acetate.They are controlled by the corresponding three structural genes (respectively by eq. SGZ, Wb said).When there is no inducer-lactose in the medium, the repressor produced by the regulatory gene binds to the operator gene, preventing or interfering with the combination of RNA polymerase and the promoter gene, so that the structural gene forms the transcription process of InR-NA If it cannot be carried out, the three enzymes necessary for lactose metabolism cannot be synthesized.When the inducer lactose exists in the medium, it will immediately combine with the repressor to change its configuration and lose its original function. At this time, the operator gene will be opened, allowing RNA polymerase to bind to the promoter gene. The transcription process of the gene to produce rnRNA and the synthesis of the three enzymes can proceed normally.Once the lactose is used up, the repressor acts again to close the channel of RNA polymerase again, so that the transcription process of structural genes stops, and the synthesis of enzymes also ends.The following figure is a schematic diagram of the operon switch.

(A) shows the process of inducible enzyme formation. (B) shows the process of repressor enzyme formation. Jacob and Monod's operon theory uses a set of regulatory control systems to explain why cells can turn on or off certain genes as needed under certain conditions.This is very helpful for us to understand how genes control the development of traits through the action of enzymes.However, it was later discovered that the operon theory does not apply to eukaryotes.Therefore, gene regulation in eukaryotes is still a subject to be studied. Prokaryotic organisms such as bacteria have a relatively simple structure. They have neither organelles nor nuclear membranes. Their genetic material—DNA or RNA—is completely exposed in the cytoplasm, and there is no dyeing structure.Therefore, in prokaryotes, the transfer of genetic information, from transcription to selection, is carried out simultaneously, and sometimes even replication, transcription and translation are trinity.Furthermore, their genes are few in number, and functionally related genes are often closely linked.So their gene regulation can be explained by operon theory.

Eukaryotes are different. They not only have various organelles, but also have nuclear membranes that surround DNA and form complex chromosomes (composed of DNA, protein and a small amount of gyA).Therefore, eukaryotes are much more complex and perfect than prokaryotes in realizing the transmission and expression of genetic information.For example, the transcription and translation of eukaryotic organisms take place in the nucleus and cytoplasm respectively. These two processes are separated in time and space, and their transcription and translation have special "machine tools". The product also needs to undergo various processing and modification before it can be transported into the cytoplasm.In addition, there are a large number of genes in eukaryotes, from fertilized eggs to complete organisms, to go through a complex process of differentiation and development, except for those genes necessary to maintain the basic life activities of cells, the genes in cells of other different tissues Always activated or repressed in different spatiotemporal sequences.Therefore, gene regulation in eukaryotes is quite complicated, including gene regulation at the chromosomal DNA level, gene regulation at the transcriptional level, and translation regulation.Biologists are currently delving into the mysteries of gene regulation in eukaryotes.

In addition, the gene regulation model reminds people that the process of organism development and cell differentiation is also regulated by genes.Modern biology has clarified that during the embryonic development of multicellular organisms, all the genes in the germ cells are copied and transmitted to each daughter cell, but most of the genes are not expressed.Which genes are expressed depends on the cell's location in the body, its developmental stage, and its environment at the time.Research in recent years has shown that there is a specific pattern of active genes (genes that are on) and inactive genes (genes that are off) in each cell, and that this pattern changes as development progresses undergo a sequence change.According to the research of Cambridge Molecular Biology Laboratory in the United Kingdom, a set of genes plays a key role in controlling the timing of cell differentiation during the embryonic development of a transparent nematode worm.They called this group of genes timing genes.Certain mutations in timing genes can alter the developmental processes of cell lineages, causing them to proceed earlier or later than normal in individuals.In addition, in 1983, researchers at the Biology Center of the University of Basel studied the embryonic development of Drosophila and found that many genes that control spatial structure in Drosophila share a common DNA (containing a unique base sequence). Call this a homology box.When a gene containing a homeobox is translated into a protein, the homeobox forms a chain of amino acids and connects to the DNA double helix.When this protein binds to the DNA of specific genes, it turns those genes on or off.If this group of genes is mutated, the structure of the adult body segment growth will be wrong, and the position where the long antennae should be grows instead.Later, some people also found sequences similar to the homology box in other organisms such as frogs, chickens, and mice.In this way, the discovery of the homeobox provides an important foothold for studying how genes regulate the development of an organism.

From this point of view, in order to gain an in-depth understanding of the mechanism of gene regulation, a very important or critical task is to figure out the original code contained in the DNA molecule or to determine the sequence of its entire acid sequence.Since the 1960s, many scientists have been engaged in this area of ​​work.For example, Phils began to study the structure of phage M& RNA in 1965, and finally figured out the whole nucleotide sequence of M& in 1975.After him, many scientists have determined the full nucleic acid sequence of bacteriophage X174DNA, as well as the full nucleic acid sequence of virus SV40 and phage fd.

In the 1980s, the analysis of the whole nucleic acid sequence of the human genome rose again.This is an international big science project.The common view now is that the human genome is estimated to have about 10,000 genes, containing 3 billion nucleic acid pairs.It is obviously not easy to determine such a huge whole nucleic acid sequence.The United States plans to spend 15 years and spend 3 billion US dollars to complete it.Once the nucleotide sequence of the human genome is clarified, people can draw a clear map of the human genome, and according to the map, predict human traits from simple DNA, analyze which genes have mutated, and What changes have taken place in the structure, on which chromosome and where the mutant gene is located, and so on.In this way, not only the genetic nature and developmental program of a person can be seen at a glance, but also how normal genes work, how abnormal genes cause diseases, and so on.No wonder some people liken the analysis of the human genome to the "Apollo" project to thoroughly understand the mysteries of the human body itself.

However, it should not be overlooked that the analysis of the human genome provides only a genetic blueprint, the possibility of trait development, and it is not possible to predict the prospects of all traits with complete reliability, because many traits (especially those related to behavior and cognition ) is determined by the interaction of genetics and the environment, and changes in the environment can cause genetically similar individuals to develop along very different paths.Therefore, an explanation of the expression of genetic information is incomplete without taking this into consideration. (Zhong Anhuan)

The past of life on earth is the course of hundreds of millions of years of evolution of living organisms.In the cells of all these living organisms lies long dormant the dense helical element of the DNA molecule, through which we can hear the echoes of history.The following short stories are examples. 1. Searching for the ancestors of mankind In Christianity and myths and legends, Adam and Eve are the ancestors of mankind.So, in real life, do human beings have a common mother?To answer this question, genetic and genomic research may be able to provide convincing evidence. As early as 1987, the British authoritative "Nature" magazine published a paper by researchers from the University of California, Berkeley. One of the conclusions of the paper is: the common mother of human beings exists, and this is "Eve".According to the investigation and calculation of scientists, Eve, the common mother of mankind, probably lived in Africa 200,000 years ago.The researchers relied on genes.Mitochondria exist in the cytoplasm of human cells, which also contain DNA, mitochondrial DNA, which is a special kind of gene.This gene can only be passed from mother to daughter.Based on this feature, researchers studied women of many ethnicities, including Africans, Asians, Caucasians, Australians, Native Americans, and New Guineans, and found that all women's mitochondrial DNA genetic profiles were in a certain segment or some are similar or identical in position.This proves that they are all related, and it is likely that their chromosomes (ie DNA) came from the same woman, and this woman is Eve in the Bible. Other researchers believe that the common mother of human beings lived in Africa 1 million years ago based on ancient corpse bones and DNA tests.Of course, further studies of human genes and genomes will go a long way toward convincingly elucidating who our common ancestor was. The second reveals the origin of the country or the nation. Who was the first colonizer of Australia and established and developed this country? The fact that is generally recognized at present is that in 1788, Captain Philip of the United Kingdom led 1044 soldiers and prisoners in Sydney Harbor on the east coast of Australia. Land and establish a colony.However, the results of recent genetic studies have revealed that Western colonists had settled in Australia in the decades before 1788. Biochemical experts from the Charles Gardner Hospital in Pas, the capital of Western Australia in Australia, traced the gene of a genetic disease and proved that the earliest Western colonists who settled in Australia were Dutch in 1712 (not as stated in the history books Carried by the British in 1788).Researchers have discovered a rare skin disease that has been traced back to 1688 by a Dutch couple living in South Africa.Since the disease is hereditary, it is plausible that only descendants of the Dutch in South Africa are likely to develop the disease.Oddly, the researchers found the disease in Aboriginal people in Western Australia.Rick Rossi, a biochemist who studies the disease, compared the genetics of the Afrikaners and Aboriginal Australians who suffered from the disease, thus confirming that their disease originated from the same ancestor: on their disease genetic map, there is a defect genes at the same location. So how to explain that these two races separated by thousands of mountains and rivers have the same disease-causing gene?There is a historical fact that can illustrate the problem.According to historical records, in 1712, the "Flagship" ship of the Dutch East India Company ran aground and sank in Shark Bay, north of the Kalmari area on the west coast of Australia. Some of them are Dutch from South Africa, and some suffer from the rare skin disease mentioned above.They bonded with the local aborigines and lived out the rest of their lives, naturally passing the disease on to the local aborigines. Another theory is that South Africa was a Dutch colony at that time, and there were many Afrikaans crew members on board the Dutch ship, some of whom suffered from this rare skin disease.From the beginning to the middle of the 18th century, many Dutch ships were wrecked on the west coast of Australia. Some people who were lucky enough to land on the Australian mainland intermarried with the local aborigines and survived. Among them, people who carried the above-mentioned defective gene inherited this rare skin disease. to their offspring.Therefore, the preliminary conclusions of genetic research conducted by Australian scientists prove that Australia's colonial history should be pushed back to 1712. 3. Get out of the misunderstanding of race theory In the mutual discrimination, massacre and even ethnic cleansing of human beings, it is often accompanied by absurd race theory to deceive people and deceive the world, as the basis for this brutal bestiality.For example, Hitler's Aryan racial superiority theory created public opinion for the brutal crime of the Nazi massacre of Jews.The argument that white people are superior to colored people is still prevalent among white people in many countries to this day. However, Professor Luca Cavalli Sforza, a human genetic researcher at Stanford University in the United States, has confirmed through nearly 50 years of genetic research: "The world is a family."The genetic differences between races are very small or very similar; and the genetic differences of each individual are far greater than the genetic differences between people of different races.In the past 50 years, Cavalli Sforza and his colleagues have collected blood samples and hairs from nearly 2,000 ethnic groups, and extracted cell genes from them for mapping analysis. Cavalli Sforza deduced genetic differences between races by profiling the genes of many races and by analyzing leukocyte surface antigens, antibodies and other proteins in the blood that are genetic markers of an individual's genetic makeup A small conclusion even reveals the genetic relationship between some races.For example, Aboriginal Australians and sub-Saharan Africans, who are similar in body shape and skin color, were previously thought to be more closely related, but genetic studies have shown that their blood classes are the furthest apart.Aboriginal Australians are more closely related to their close neighbors, Southeast Asians.The difference in appearance between Europeans and Africans is only the result of their respective adaptations to the local climate after migration. For many years, Europeans have believed that white people are superior to all races, relying on the fact that they almost conquered the whole world in the colonial era.However, Cavalli Sforza's genetic research disproves this theory.Studies have shown that 65% of the genes of European Caucasians come from Asians, and 35% come from Africans, which is a hybrid race of Asian and African races.Therefore, European Caucasians are Asian and African.The same blood runs in the veins of Caucasians and Afro-Asians. Cavalli Sforza's genetic research also proves that Africa is the birthplace of human beings and the starting point of global migration. The reason for the difference between today's Africans and other races is that the different lives of the ancient ages have led to the variation of people's genes.This is the same conclusion as the above-mentioned common mother of mankind originated from Africa. 4. Tracing the origin of the disease No matter how dangerous and intractable the disease is, as long as the cause of the disease is found, it will lay the foundation for conquering the disease.Tracing the root of the disease is one of the effective ways to find the cause.Genetic research can effectively find the root causes of diseases, especially genetic diseases. One day in September 1495, in a small and beautiful village near the town of Vierreevroy on the Strait of Calais, France, the church bell rang sadly and sadly.People from the village gathered at the church for the funeral of an old couple named Geoffrey and Mary.However, people don't think that the Sri Lankan people are gone, but they have left a causative gene of a genetic disease to future generations. Long years passed. In May 1991, André Chaventry, a researcher at the French National Institute of Demographic Statistics, was invited by Professor Avery of the Paris Psychiatric Hospital to investigate the incidence of manic-depressive psychosis.Shortly after the two collaborated on research, they discovered that a certain gene segment of DNA was not only involved in mental illness, but also glaucoma and diabetes.Diabetes and glaucoma were also more common in family members with mental illness than in the general population.During the investigation, the researchers found a particularly striking family line, dating back to the 15th century, whose ancestors were Geoffrey and Mary.The researchers went to the family genetic line recorded in the church, that is, the family tree, and through gene tracing analysis, with the cooperation of ophthalmologists, they proved that the Jeffreys had glaucoma and inherited the glaucoma-causing gene. .More than 100 of their descendants suffered from glaucoma over the centuries, and thousands of others carried the glaucoma-causing gene, but did not show symptoms.Finally, the researchers determined the location of glaucoma on the chromosome through genetic analysis.This laid the foundation for future prevention and treatment of glaucoma. 5. Understanding differences in ethnic groups and diseases Gene research is now playing an increasingly important role in revealing similarities and differences in ethnic differences and diseases.When it is difficult for people to identify an ethnicity from aspects such as history, place of residence, and language, genetic research becomes an important basis for judgment. In the past, anthropologists believed that the Kwasan people of South Africa might be one of the oldest races of human beings, because of the breath-taking speech in their language, which led linguists to believe that they were the direct descendants of the most primitive ancestors of human beings. descendants.However, genetic research by Stanford University in the United States shows that the Kwasan people are a very ancient mixed race of West Asians and Africans, and their fusion sites are Ethiopia and the Middle East, which shows that the Kwasan people are not one of the oldest races of mankind . The genetic analysis of Native Americans by scholars at Stanford University in the United States also showed that the three types of Natives have different genes, and their languages ​​are also different.The Indians who dominate in North and South America have only O blood type (blood type can be used as a sign of genetic classification); There is also type A blood; the Inuit in Alaska and Canada, like people in other parts of the world, have the four blood types A, B, O, and AB.This can explain that they are Asians who settled in America in different periods. Likewise, for populations where it is difficult to discern origin and ethnicity, such as the Indian Ocean.The short blacks in the Malay Peninsula and the Philippines can also be classified into corresponding ethnic groups through genetic analysis in the future. Gene and genome projects have also made major contributions in helping to understand the identities and differences in human disease.Tracing the origin of genetic diseases mentioned above can be regarded as the identity of the disease, while the difference of the disease-causing gene results in the vastly different diseases and different groups of people who are susceptible to a certain disease.For example, Mongolians rarely suffer from scarlet fever; thalassemia has the highest incidence rate among the Dai and Jingpo ethnic groups in Yunnan, China, about 5.51%; the incidence rate of nasopharyngeal carcinoma in Chinese is relatively high; .In addition to the objective environment of onset, the above-mentioned diseases (including many diseases) can find differences in the genetic loci of different ethnic groups, that is, the etiology, which lays the foundation for future clinical diagnosis and treatment. (Zhang Tianqin) "Cloning" is transliterated from the English "Chlone", which means asexual reproduction.It has a different level of meaning in the field of biology. 1. DNA cloning is also called molecular cloning, which means that a specific DNA fragment is inserted into a vector (such as plasmid and virus, etc.) The "group" of the fragment. 2.Cell clone refers to a population of cells formed by the division of a single common ancestor cell.For example, a group of cells with the same genetic background formed by dividing a single cell for several generations in a culture medium in vitro is a cell clone.As another example, in vertebrates, when foreign substances (such as bacteria or viruses) invade, specific recognition antibodies will be produced through immune responses.All the plasma cells that produce a particular antibody are derived from the same B cell division, and such a population of plasma cells is also a cell clone. 3.Individual cloning refers to a population of two or more individuals that are genetically identical.For example, two identical twins are a clone!Because they come from the same fertilized egg cell, the genetic background is exactly the same; an animal or plant with the same genetic background obtained through nuclear transfer is also a clone. For example, in 1998, British scientists transplanted the nucleus of mouse cumulus cells to remove After the nucleus of the oocyte was obtained, more than 20 fully developed mice were obtained. These mouse populations are a clone, and the carrot population developed from more than two somatic cells in a carrot is also a clone. , because their genetic backgrounds are identical.What is familiar to everyone and the most well-known British scientist about "cloning" is the team led by British scientist Wilmut, who successfully "duplicated" the first sheep "Dolly" in 1997 by using cloning technology. . Dr Ian Wilmut is an embryologist at the Roslin Institute in Edinburgh, UK.He was born in Hampton Luce, near Warwick, a city in central England, in 1945. He studied at the University of Nottingham, and his tutor was Eric Lamin, a world-renowned reproductive expert.After graduation, he entered the field of embryology and has been engaged in the research of animal genetic technology. In 1971, he went to Darwin College of Cambridge University for advanced studies, and obtained a doctorate two years later. The title of his doctoral thesis was "On the Freezing Technology of Pig Semen".After graduation, he went to the Roslyn Institute of Animal Reproduction in Edinburgh, Scotland.The Institute is an independent animal research institution jointly funded by the government and Edinburgh Pharmaceutical Protein Co., Ltd., which gradually evolved into the Roslin Institute. Dr. Wilmut has been working in reproductive science for more than 20 years. The first calf was bred from frozen embryos in 1973.A cow can usually produce 5 to 10 calves in her lifetime.By freezing embryos from the best meat and milk quality cows and implanting them in other cows after thawing, Dr. Wilmut has enabled cattle farmers to greatly improve the quality of their cattle. In 1986, while attending a conference in Ireland, Dr. Wilmut overheard people talking in a bar about a scientist who had grown a sheep from an embryo that had already developed, convincing him that it was possible to clone large livestock. Finally, Dr. Wilmut led a team of 12 scientists to complete a scientific research project that attracted worldwide attention. Mammals generally reproduce sexually.Mammalian eggs are first produced from oogonia in the ovary. Oogonia have double the genetic material and are diploid cells.It divides several times and eventually becomes a mature egg cell that is haploid (only half of the chromosomes in the body cell).However, it is impossible for this egg cell to develop into a new individual. It must be fertilized (combined with a sperm containing only haploid chromosomes) and become a diploid fertilized egg again in order to continue to develop and form a new life. The cultivation of "cloned sheep" is the same as the cultivation process of cloning other mammals. First, mature egg cells must be obtained.Today, scientists have adopted "superovulation technology" to inject pregnant horse serum gonadotropin and human chorionic gonadotropin into adult ewes.This allows more eggs to mature and be released at one time in their ovaries.When ovulation occurs, the staff can remove this mature egg cell through surgery or laparoscopy. Egg cells are composed of two parts, the nucleus and the cytoplasm.Egg cells are very small, generally only between 80 and 100 microns. When operating, scientific workers must rely on the help of an injector, under the condition of tens of times of magnification, use a special ultra-thin glass tube to insert into the egg, and suck out the nucleus of the egg.The egg becomes an anucleated cell (the egg has no nuclear genetic material).Then "nuclear transfer" is carried out. Generally, the nuclei used for nuclear transfer are mostly the nuclei of embryonic blastomeres (each nucleus of a blastomere has the ability to divide and proliferate).However, the individual obtained by separating and cutting such cells (or embryos) cannot be called a "cloned individual". why?One is that "Dolly" does not use the nucleus of embryonic cells, but the nucleus of "somatic cells (mammary gland cells) to carry out nuclear transfer, divide and develop into new individuals. According to the point of view of developmental biology, adult somatic cells It is a "directed" and differentiated cell in a certain program, that is, the nature of this cell has been finalized, which type of cell or tissue is that type of cell or tissue, just as mammary gland cells can only develop into mammary glands Like tissues, it is impossible to "turn back" and regain "totipotency". However, even if the somatic cells of "Dolly" have "clear direction", under certain conditions, they still have "totipotency". The second is that the somatic cells that are transferred into the eggs not only contain doubled chromosomes, but also the chromosomes of the resulting offspring cells are the genetic copies of the somatic cells, so the genetic properties of the individuals developed from this are the same as those of the nuclear donor. parents are identical. Here is an overview of the birth process of "Dolly": a piece of ordinary cell tissue that has no reproductive function was extracted from the mammary gland of a 6-year-old Sett ewe in Finland, and cultured for 6 days under special conditions to make these The nucleus of the cell enters the dormancy period; the genetic material of an unfertilized egg is removed by micromanipulation; the nucleus of the mammary gland cell is introduced into the egg from which the nucleus has been removed through cell fusion to form a recombinant embryo; the recombinant embryo is transferred to After a few days of in vivo development in the oviduct of a suitable donor sheep, well-developed embryos are taken out from the oviduct, and then transplanted into the uterus of a suitable recipient ewe, and finally lambs are born from it. We can see from the production process of "Dolly" that it has not undergone the fertilization process of sperm and egg cells, and belongs to asexual reproduction, so it is called "cloned sheep". The wonderful name "Dolly" was borrowed by Wilmut from his favorite country singer, Dolly Parton. There are many plants and animals that use asexual reproduction in daily life. For example, the offspring produced by plant fiber cutting, grafting, and tuber propagation are all clones. Under natural conditions, because many plants are inherently suitable for vegetative reproduction, they are easy to clone.In animals, this method of asexual reproduction is more common in invertebrates, such as protozoan fission and so on.However, for higher animals, they generally can only reproduce sexually in their natural state. If they are to reproduce asexually, scientists must go through a series of complicated procedures. The current methods of cloning mammals are as follows from simple to complex: (1) Embryo segmentation. Embryo segmentation is considered "cloning" in many countries, but it is not strictly cloning.The unimplanted early embryos are divided into two by microsurgical methods, divided into four or more times, and then transplanted into the recipients respectively to allow them to conceive and give birth.More than two offspring can be cloned from one embryo, and the genetic performance is exactly the same.At present, mice and rabbits have been cloned by embryo segmentation.Goats, sheep, pigs, cattle and horses etc. (2) Embryo cell nuclear transfer. Embryo nuclear transfer technology is a step forward than embryo segmentation technology.It uses microsurgical methods to separate early embryonic cells for implantation, and introduces a single cell into unfertilized mature oocytes that have had their chromosomes removed. After electrofusion, the cytoplasm of the oocyte fuses with the nucleus of the introduced embryo , divide, and develop into embryos.The embryo is transferred to the recipient, who is allowed to conceive and give birth.The currently known animals cloned by embryonic cell nuclear transfer include mice, rabbits, goats, sheep, and pigs.Cows and monkeys etc. (3) Embryonic stem cell nuclear transfer. The embryonic or fetal primordial germ cells are suppressed and differentiated, so that the number of cells is multiplied, but the cells are not differentiated, and each cell still has the ability to develop into an individual.Using the above-mentioned nuclear transfer technology, the single cell is introduced into the mature oocyte from which the chromosome has been removed to clone the embryo, and after transplantation to the recipient, pregnancy, litter, and cloned animals are produced. (4) Embryo chimerism. Two embryonic cells (same or heterogeneous animal embryos) are chimerized and developed together into one embryo, which is called a chimeric embryo.The embryo is then transferred to a recipient for pregnancy and offspring.If the litter has cells of the embryos of the above two animals, it is called a chimeric animal.For example, the embryonic cells of black mice and white mice of the same kind were chimerized, and black and white mice were born.Different species of sheep and goat embryo cells chimerism can give birth to sheep and goats, which have the characteristics of both sheep and goats.At present, chimera animals include mice, rats, sheep, goats, pigs, and cattle; interspecies chimera animals include rat-mouse chimera, sheep-goat chimera, horse-zebra chimera, and cow-buffalo chimera . (5) Somatic cell nuclear transfer. Animal somatic cells are suppressed and cultured, so that the cells are in a dormant state.Using the above method of nuclear transfer, it is introduced into the mature oocyte cloned embryo from which the chromosome has been removed, and the recipient is transplanted, pregnant and littered, and the animal is cloned.Like the cloned sheep Dolly.It takes out an ordinary cell that has no reproductive function from the mammary gland of an adult ewe, and separates the gene of this cell for use; The gene is taken out and replaced with the gene of the first ewe mammary gland cell, and this gene is activated by the discharge of the egg cell that has been "swapped" to make it start cell division like a normal fertilized egg; when the cell division progresses to a certain stage, After the embryo had formed, the embryo was implanted into a third ewe, which gave birth to "Dolly" after a normal pregnancy.This technology is currently the only one that has been successful. The birth of cloned sheep is a milestone in the history of bioengineering technology.It marks the early arrival of the century of biology.The advent of the cloned sheep "Dolly" has broken through the traditional method of using embryonic cells for nuclear transfer, allowing scientists to have a new and very effective technology to deeply study a series of important biological issues, both theoretically and practically are of great significance. (Zhao Xueshu) Genetic engineering is a technology for artificially cutting, recombining, transferring and expressing genes, and it is an artificial intervention on biological inheritance at the molecular level. In 1973, Professor Cohen of Stanford University in the United States took out two different materials from E. coli.Each of them has an antibiotic drug gene, which is "cut out", and then two genes are "cut out", and then these two genes are "spliced" into the same plasmid.The new plasmid is called a "hybrid plasmid".When this hybrid plasmid enters E. coli, these E. coli can resist both drugs, and the offspring of this E. coli are all dual drug resistant. This means that the "hybrid material" is also capable of self-replication during E. coli cell division.It marked the first triumph of genetic engineering. 1974年，科恩又把金黄色葡萄球菌的质球（上面具有抗青霉素的基因）和大肠杆菌的质粒“组装”成杂合质粒，送入大肠杆菌体内，使这种大肠杆菌获得了对青霉素的抗药性。这说明，金黄色葡萄球菌质粒上的抗青霉素基因，由杂合质粒带到大杆菌体内，更重要的是表明外来基因在大肠杆菌体内同样也发生作用（专业上称为表达）。 科思又将非洲爪赠的DNA与大肠杆菌的质粒“拼接”，获得成功，拼接后的杂合质粒进入大肠杆菌，产生了非洲爪赠的核糖体核糖核酸（币W人。两栖动物的基因能在细菌里发挥作用，也能在细菌里不断复制的事实说明，基因工程完全可以不受生物种类的限制，而按照人类的意愿去拼接基因，创造新的生物。 科恩随后以DNAlifl技术发明人的身份向美国专利局申报了世界上第一个基因工程的技术专利。科恩的实验首次打破了不同物种在亿万年中形成的天然屏障，他的成功标志着任何不同种类生物学基因都能通过基因工程技术重组到一起，人类可以根据自己的意愿定向地改造生物的遗传特性，甚至创造新的生命类型。科恩获得专利技术的消息引起了全球轰动，在短短几年中，世界上许多国家的上百个实验室开展了基因工程的研究。 随着科思及其同事利用重组DNA技术从哺乳动物基因组中切割了一个基因，植入大肠杆菌获得成功后。投资家鲍勃·斯旺森说服博耶成立遗传技术公司——世界上第一家利用重组DNA技术制造蛋白质用于治疗人体疾病的公司，它于20世纪70年代在美国诞生，生物工程从此步入产业化。 基因工程一般包括四个方面的基本内容：一是取得符合人们的要求的DNA片段，这种DNA片段被为“目的基因”；二是将目的基因与质粒或病毒DNA连接成重组DNA（质粒和病毒DNA称作载体）；三是把重组DNA引入某种细胞（称为受体细胞）；四是把目的基因能表达的受体细胞挑选出来。DNA分子很小，其直径只有20埃，约相当于五百万分之一厘米，在它们身上进行“手术”是非常困难的，因此基因工程实际上是一种“超级显微工程”，对——的切割、缝合与转运，必须有特殊的工具。首先，要把所需基因——目的基因从供体DNA长链中准确地剪切下来。1968年，沃纳·阿尔伯博士、丹尼尔·内森斯博士和汉密尔·史密斯博士第一次从大肠杆菌中提取出了限制性内切酶能够在DNA上寻找特定的“切点”，认准后将DNA分子的双链交错地切断。人们把这种限制性内切酶称为“分子剪刀”。这种“分子剪刀”可以完整地切下个别基因。自70年代以来，人们已经分离提取了400多种“分子剪刀”，其中许多“分子剪刀”的特定识别切点已被弄清。有了形形色色的“分子剪刀”，人们就可以随心所欲地进行DNA分子长链的切割了。由于限制性内切酶的发现，阿尔伯、史密斯和内森斯共享1978年诺贝尔生理和医学奖。 DNA的分子链切开后，还得缝接起来以完成基因的拼接。1976年，科学家们在5个实验室里几乎同时发现并提取出一种酶，这种酶可以将两个DNA片段连接起来，修复好DNA铁的断裂口。1974年以后，科学界正式肯定了这一发现，并把这种酶叫做DNA连接酶。 从此，DNA连接酶就成了名副其实的“缝合”基因的“分子针线”。只要在用同一种“分子剪刀”剪切的两种DNA碎片中加上“分子针线”，就会把两种DNA片段重新连接起来。 把“拼接”好的DNA分子运送到受体细胞中去，必须寻找一种分子小、能自由进出细胞，而且在装载了外来的的管DNA片段后仍能照样复制的运载体。 基因的理想运载工具是病毒和噬菌体，病毒不仅在同种生物之间，甚至可以在人和兔培养细菌细胞转移。还有一种理想的载体是质粒。质粒能自由进出细菌细胞，当用“分子剪刀”把它切开，再给它安装上一段外来的DNA片段后，它依然如故地能自我复制。因此，它是一种理想的运载体。有了限制性内切酶、连接酶及运载体，进行基因工程就可以如愿以偿了。 把目的基因装在运载体上，运载体将目的基因运到受体细胞是基因工程的最后一步。 一般情况下，转化成功率为百万分之一。为此，遗传工程师们创造了低温条件下用氯化钙处理受体细胞和增加重组DNA浓度的办法来提高转化率。采用氯化钙处理后，能增大体细胞的细胞壁透性，从而使杂种DNA分子更容易进入。目的基因的导人过程是肉眼看不到的。因此，要知道导人是否成功，事先应找到特定的标志。例如我们用一种经过改造的抗四环素质粒PSC100作载体，将一种基因移入自身无抗性的大肠杆菌时，如果基因移入后大肠杆菌不能被四环素杀死，就说明转入获得成功了。