Category Archives: Cloning

Abstract

There are, in mankind, two kinds of heredity: biological and cultural. Cultural inheritance makes possible for humans what no other organism can accomplish: the cumulative transmission of experience from generation to generation. In turn, cultural inheritance leads to cultural evolution, the prevailing mode of human adaptation. For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments. Nevertheless, natural selection persists in modern humans, both as differential mortality and as differential fertility, although its intensity may decrease in the future. More than 2,000 human diseases and abnormalities have a genetic causation. Health care and the increasing feasibility of genetic therapy will, although slowly, augment the future incidence of hereditary ailments. Germ-line gene therapy could halt this increase, but at present, it is not technically feasible. The proposal to enhance the human genetic endowment by genetic cloning of eminent individuals is not warranted. Genomes can be cloned; individuals cannot. In the future, therapeutic cloning will bring enhanced possibilities for organ transplantation, nerve cells and tissue healing, and other health benefits.

Chimpanzees are the closest relatives of Homo sapiens, our species. There is a precise correspondence bone by bone between the skeletons of a chimpanzee and a human. Humans bear young like apes and other mammals. Humans have organs and limbs similar to birds, reptiles, and amphibians; these similarities reflect the common evolutionary origin of vertebrates. However, it does not take much reflection to notice the distinct uniqueness of our species. Conspicuous anatomical differences between humans and apes include bipedal gait and an enlarged brain. Much more conspicuous than the anatomical differences are the distinct behaviors and institutions. Humans have symbolic language, elaborate social and political institutions, codes of law, literature and art, ethics, and religion; humans build roads and cities, travel by motorcars, ships, and airplanes, and communicate by means of telephones, computers, and televisions.

The hominin lineage diverged from the chimpanzee lineage 67 Ma, and it evolved exclusively in the African continent until the emergence of Homo erectus, somewhat before 1.8 Ma. Shortly after its emergence in tropical or subtropical Africa, H. erectus spread to other continents. Fossil remains of H. erectus (sensu lato) are known from Africa, Indonesia (Java), China, the Middle East, and Europe. H. erectus fossils from Java have been dated at 1.81 0.04 and 1.66 0.04 Ma and from Georgia at 1.61.8 Ma (1). Anatomically distinctive H. erectus fossils have been found in Spain, deposited before 780,000 y ago, the oldest in southern Europe (2).

The transition from H. erectus to H. sapiens occurred around 400,000 y ago, although this date is not well determined owing to uncertainty as to whether some fossils are erectus or archaic forms of sapiens. H. erectus persisted for some time in Asia, until 250,000 y ago in China and perhaps until 100,000 ago in Java, and thus was contemporary with early members of its descendant species, H. sapiens. Fossil remains of Neandertal hominids (Homo neanderthalensis), with brains as large as those of H. sapiens, appeared in Europe earlier than 200,000 y ago and persisted until 30,000 or 40,000 y ago (3, 4).

There is controversy about the origin of modern humans. Some anthropologists argue that the transition from H. erectus to archaic H. sapiens and later to anatomically modern humans occurred consonantly in various parts of the Old World. Proponents of this multiregional model emphasize fossil evidence showing regional continuity in the transition from H. erectus to archaic and then modern H. sapiens. Most anthropologists argue instead that modern humans first arose in Africa somewhat before 100,000 y ago and from there spread throughout the world, eventually replacing elsewhere the preexisting populations of H. erectus, H. neanderthalensis, and archaic H. sapiens. The African origin of modern humans is supported by a wealth of recent genetic evidence and is therefore favored by many evolutionists (2, 4).

We know about these matters in three ways: by comparing living primates, including humans, with each other; by discovery and investigation of fossil remains of primates that lived in the past; and by comparing their DNA, proteins, and other molecules. DNA and proteins give us the best information about how closely related we are to each of the primates and those to each other. However, to know how the human lineage changed in anatomy and behavior over time as our ancestors became more and more human-like, we have to study fossils and the tools they used and made, as well as other remnants of their activities (2, 5).

Humans live in groups that are socially organized and so do other primates. However, other primate societies do not approach the complexity of human social organization. A distinctive human social trait is culture, which may be understood as the set of nonstrictly biological human activities and creations. Culture includes social and political institutions, ways of doing things, religious and ethical traditions, language, common sense and scientific knowledge, art and literature, technology, and in general all of the creations of the human mind. The advent of culture has brought with it cultural evolution, a superorganic mode of evolution superimposed on the organic mode, that has become the dominant mode of human evolution. Cultural evolution has come about because of cultural inheritance, a distinctively human mode of achieving adaptation to the environment (2, 6, 7).

There are in mankind two kinds of heredity: the biological and the cultural. Biological inheritance in humans is very much like that in any other sexually reproducing organism; it is based on the transmission of genetic information encoded in DNA from one generation to the next by means of the sex cells. Cultural inheritance, on the other hand, is based on transmission of information by a teaching-learning process, which is in principle independent of biological parentage. Culture is transmitted by instruction and learning, by example and imitation, through books, newspapers, radio, television, and motion pictures, through works of art, and through any other means of communication. Culture is acquired by every person from parents, relatives, and neighbors and from the whole human environment. Acquired cultural traits may be beneficial but also toxic; for example, racial prejudice or religious bigotry.

Biological heredity is Mendelian or vertical; it is transmitted from parents to their children, and only inherited traits can be transmitted to the progeny. (New mutations are insignificant in the present context.) Cultural heredity is Lamarckian: acquired characters can be transmitted to the progeny. However, cultural heredity goes beyond Lamarckian heredity, because it is horizontal and oblique and not only vertical. Traits can be acquired from and transmitted to other members of the same generation, whether or not they are relatives, and also from and to all other individuals with whom a person has contact, whether they are from the same or from any previous or ensuing generation.

Cultural inheritance makes possible for people what no other organism can accomplishthe cumulative transmission of experience from generation to generation. Animals can learn from experience, but they do not transmit their experiences or their discoveries (at least not to any large extent) to the following generations. Animals have individual memory, but they do not have a social memory. Humans, on the other hand, have developed a culture because they can transmit cumulatively their experiences from generation to generation.

Cultural inheritance makes possible cultural evolution, a new mode of adaptation to the environment that is not available to nonhuman organisms. Organisms in general adapt to the environment by means of natural selection, by changing over generations their genetic constitution to suit the demands of the environment. However, humans, and humans alone, can also adapt by changing the environment to suit the needs of their genes. (Animals build nests and modify their environment also in other ways, but the manipulation of the environment by any nonhuman species is trivial compared with mankind's manipulation.) For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments.

To extend its geographical habitat, or to survive in a changing environment, a population of organisms must become adapted, through slow accumulation of genetic variants sorted out by natural selection, to the new climatic conditions, different sources of food, different competitors, and so on. The discovery of fire and the use of shelter and clothing allowed humans to spread from the warm tropical and subtropical regions of the Old World to the whole Earth, except for the frozen wastes of Antarctica, without the anatomical development of fur or hair. Humans did not wait for genetic mutants promoting wing development; they have conquered the air in a somewhat more efficient and versatile way by building flying machines. People travel the rivers and the seas without gills or fins. The exploration of outer space has started without waiting for mutations providing humans with the ability to breathe with low oxygen pressures or to function in the absence of gravity; astronauts carry their own oxygen and specially equipped pressure suits. From their obscure beginnings in Africa, humans have become the most widespread and abundant species of mammal on earth. It was the appearance of culture as a superorganic form of adaptation that made mankind the most successful animal species.

Cultural adaptation has prevailed in mankind over biological adaptation because it is a more effective mode of adaptation; it is more rapid and it can be directed. A favorable genetic mutation newly arisen in an individual can be transmitted to a sizeable part of the human species only through innumerable generations. However, a new scientific discovery or technical achievement can be transmitted to the whole of mankind, potentially at least, in less than one generation. Witness the rapid spread of personal computers, iPhones, and the Internet. Moreover, whenever a need arises, culture can directly pursue the appropriate changes to meet the challenge. On the contrary, biological adaptation depends on the accidental availability of a favorable mutation, or of a combination of several mutations, at the time and place where the need arises (2, 6, 7).

There is no scientific basis to the claim sometimes made that the biological evolution of mankind has stopped, or nearly so, at least in technologically advanced countries. It is asserted that the progress of medicine, hygiene, and nutrition have largely eliminated death before middle age; that is, most people live beyond reproductive age, after which death is inconsequential for natural selection. That mankind continues to evolve biologically can be shown because the necessary and sufficient conditions for biological evolution persist. These conditions are genetic variability and differential reproduction. There is a wealth of genetic variation in mankind. With the trivial exception of identical twins, developed from a single fertilized egg, no two people who live now, lived in the past, or will live in the future, are likely to be genetically identical. Much of this variation is relevant to natural selection (5, 8, 9).

Natural selection is simply differential reproduction of alternative genetic variants. Natural selection will occur in mankind if the carriers of some genotypes are likely to leave more descendants than the carriers of other genotypes. Natural selection consists of two main components: differential mortality and differential fertility; both persist in modern mankind, although the intensity of selection due to postnatal mortality has been somewhat attenuated.

Death may occur between conception and birth (prenatal) or after birth (postnatal). The proportion of prenatal deaths is not well known. Death during the early weeks of embryonic development may go totally undetected. However, it is known that no less than 20% of all ascertained human conceptions end in spontaneous abortion during the first 2 mo of pregnancy. Such deaths are often due to deleterious genetic constitutions, and thus they have a selective effect in the population. The intensity of this form of selection has not changed substantially in modern mankind, although it has been slightly reduced with respect to a few genes such as those involved in Rh blood group incompatibility.

Postnatal mortality has been considerably reduced in recent times in technologically advanced countries. For example, in the United States, somewhat less than 50% of those born in 1840 survived to age 45, whereas the average life expectancy for people born in the United States in 1960 is 78 y (Table 1) (8, 10). In some regions of the world, postnatal mortality remains quite high, although there it has also generally decreased in recent decades. Mortality before the end of reproductive age, particularly where it has been considerably reduced, is largely associated with genetic defects, and thus it has a favorable selective effect in human populations. Several thousand genetic variants are known that cause diseases and malformations in humans; such variants are kept at low frequencies due to natural selection.

Percent of Americans born between 1840 and 1960 surviving to ages 15 and 45

It might seem at first that selection due to differential fertility has been considerably reduced in industrial countries as a consequence of the reduction in the average number of children per family that has taken place. However, this is not so. The intensity of fertility selection depends not on the mean number of children per family, but on the variance in the number of children per family. It is clear why this should be so. Assume that all people of reproductive age marry and that all have exactly the same number of children. In this case, there would not be fertility selection whether couples all had very few or all had very many children. Assume, on the other hand, that the mean number of children per family is low, but some families have no children at all or very few, whereas others have many. In this case, there would be considerable opportunity for selectionthe genotypes of parents producing many children would increase in frequency at the expense of those having few or none. Studies of human populations have shown that the opportunity for natural selection often increases as the mean number of children decreases. An extensive study published years ago showed that the index of opportunity for selection due to fertility was four times larger among United States women born in the 20th century, with an average of less than three children per woman, than among women in the Gold Coast of Africa or in rural Quebec, who had three times or more children on average (Table 2) (8, 11). There is no evidence that natural selection due to fertility has decreased in modern human populations.

Mean number of children per family and index of opportunity for fertility selection If, in various human populations

Natural selection may decrease in intensity in the future, but it will not disappear altogether. As long as there is genetic variation and the carriers of some genotypes are more likely to reproduce than others, natural selection will continue operating in human populations. Cultural changes, such as the development of agriculture, migration from the country to the cities, environmental pollution, and many others, create new selective pressures. The pressures of city life are partly responsible for the high incidence of mental disorders in certain human societies. The point to bear in mind is that human environments are changing faster than ever owing precisely to the accelerating rate of cultural change, and environmental changes create new selective pressures, thus fueling biological evolution.

Natural selection is the process of differential reproduction of alternative genetic variants. In terms of single genes, variation occurs when two or more alleles are present in the population at a given gene locus. How much genetic variation exists in the current human population? The answer is quite a lot, as will be presently shown, but natural selection will take place only if the alleles of a particular gene have different effects on fitness; that is, if alternative alleles differentially impact the probability of survival and reproduction.

The two genomes that we inherit from each parent are estimated to differ at about one or two nucleotides per thousand. The human genome consists of somewhat more than 3 billion nucleotides (12). Thus, about 36 million nucleotides are different between the two genomes of each human individual, which is a lot of genetic polymorphism. Moreover, the process of mutation introduces new variation in any population every generation. The rate of mutation in the human genome is estimated to be about 108, which is one nucleotide mutation for every hundred million nucleotides, or about 30 new mutations per genome per generation. Thus, every human has about 60 new mutations (30 in each genome) that were not present in the parents. If we consider the total human population, that is 60 mutations per person multiplied by 7 billion people, which is about 420 billion new mutations per generation that are added to the preexisting 36 million polymorphic nucleotides per individual.

That is a lot of mutations, even if many are redundant. Moreover, we must remember that the polymorphisms that count for natural selection are those that impact the probability of survival and reproduction of their carriers. Otherwise, the variant nucleotides may increase or decrease in frequency by chance, a process that evolutionists call genetic drift, but will not be impacted by natural selection (2, 12, 13).

More than 2,000 human diseases and abnormalities that have a genetic causation have been identified in the human population. Genetic disorders may be dominant, recessive, multifactorial, or chromosomal. Dominant disorders are caused by the presence of a single copy of the defective allele, so that the disorder is expressed in heterozygous individuals: those having one normal and one defective allele. In recessive disorders, the defective allele must be present in both alleles, that is, it is inherited from each parent to be expressed. Multifactorial disorders are caused by interaction among several gene loci; chromosomal disorders are due to the presence or absence of a full chromosome or a fragment of a chromosome (14, 15).

Examples of dominant disorders are some forms of retinoblastoma and other kinds of blindness, achondroplastic dwarfism, and Marfan syndrome (which is thought to have affected President Lincoln). Examples of recessive disorders are cystic fibrosis, Tay-Sachs disease, and sickle cell anemia (caused by an allele that in heterozygous condition protects against malaria). Examples of multifactorial diseases are spina bifida and cleft palate. Among the most common chromosomal disorders are Down syndrome, caused by the presence of an extra chromosome 21, and various kinds due to the absence of one sex chromosome or the presence of an extra one, beyond the normal condition of XX for women and XY for men. Examples are Turners syndrome (XO) and Klinefelters syndrome (XXY) (16).

The incidence of genetic disorders expressed in the living human population is estimated to be no less than 2.56%, impacting about 180 million people. Natural selection reduces the incidence of the genes causing disease, more effectively in the case of dominant disorders, where all carriers of the gene will express the disease, than for recessive disorders, which are expressed only in homozygous individuals. Consider, for example, phenylketonuria (PKU), a lethal disease if untreated, due to homozygosis for a recessive gene, which has an incidence of 1 in 10,000 newborns or 0.01%. PKU is due to an inability to metabolize the amino acid phenylalanine with devastating mental and physical effects. A very elaborate diet free of phenylalanine allows the patient to survive and reproduce if started early in life. The frequency of the PKU allele is about 1%, so that in heterozygous conditions it is present in more than 100 million people, but only the 0.01% of people who are homozygous express the disease and are subject to natural selection. The reduction of genetic disorders due to natural selection is balanced with their increase due to the incidence of new mutations.

Lets consider another example. Hereditary retinoblastoma is a disease attributed to a dominant mutation of the gene coding for the retinoblastoma protein, RB1, but it is actually due to a deletion in chromosome 13. The unfortunate child with this condition develops a tumorous growth during infancy that, without treatment, starts in one eye and often extends to the other eye and then to the brain, causing death before puberty. Surgical treatment now makes it possible to save the life of the child if the condition is detected sufficiently early, although often one or both eyes may be lost. The treated person can live a more or less normal life, marry, and procreate. However, because the genetic determination is dominant (a gene deletion), one half of the progeny will, on the average, be born with the same genetic condition and will have to be treated. Before modern medicine, every mutation for retinoblastoma arising in the human population was eliminated from the population in the same generation owing to the death of its carrier. With surgical treatment, the mutant condition can be preserved, and new mutations arising each generation are added to those arisen in the past (refs. 17 and 18; http://www.abedia.com/wiley/index.html).

The proportion of individuals affected by any one serious hereditary infirmity is relatively small, but there are more than 2,000 known serious physical infirmities determined by genes. When all these hereditary ailments are considered together, the proportion of persons born who will suffer from a serious handicap during their lifetimes owing to their heredity is more than 2% of the total population, as pointed out above (refs. 15, 16, and 19; http://www.abedia.com/wiley/index.html).

The problem becomes more serious when mental defects are taken into consideration. More than 2% of the population is affected by schizophrenia or a related condition known as schizoid disease, ailments that may be in some cases determined by a single mutant gene. Another 3% or so of the population suffer from mild mental retardation (IQ less than 70). More than 100 million people in the world suffer from mental impairments due in good part to the genetic endowment they inherited from their parents.

Natural selection also acts on a multitude of genes that do not cause disease. Genes impact skin pigmentation, hair color and configuration, height, muscle strength and body shape, and many other anatomical polymorphisms that are apparent, as well as many that are not externally obvious, such as variations in the blood groups, in the immune system, and in the heart, liver, kidney, pancreas, and other organs. It is not always known how natural selection impacts these traits, but surely it does and does it differently in different parts of the world or at different times, as a consequence of the development of new vaccines, drugs, and medical treatments, and also as a consequence of changes in lifestyle, such as the reduction of the number of smokers or the increase in the rate of obesity in a particular country.

Where is human evolution going? Biological evolution is directed by natural selection, which is not a benevolent force guiding evolution toward sure success. Natural selection brings about genetic changes that often appear purposeful because they are dictated by the requirements of the environment. The end result may, nevertheless, be extinctionmore than 99.9% of all species that ever existed have become extinct. Natural selection has no purpose; humans alone have purposes and they alone may introduce them into their evolution. No species before mankind could select its evolutionary destiny; mankind possesses techniques to do so, and more powerful techniques for directed genetic change are becoming available. Because we are self-aware, we cannot refrain from asking what lies ahead, and because we are ethical beings, we must choose between alternative courses of action, some of which may appear as good and others as bad.

The argument has been advanced that the biological endowment of mankind is rapidly deteriorating owing precisely to the improving conditions of life and to the increasing power of modern medicine. The detailed arguments that support this contention involve some mathematical exercises, but their essence can be simply presented. Genetic changes (i.e., point or chromosome mutations) arise spontaneously in humans and in other living species. The great majority of newly arising mutations are either neutral or harmful to their carriers; only a very small fraction are likely to be beneficial. In a human population under the so-called natural conditions, that is, without the intervention of modern medicine and technology, the newly arising harmful mutations are eliminated from the population more or less rapidly depending on how harmful they are. The more harmful the effect of a mutation, the more rapidly it will be eliminated from the population by the process of natural selection. However, owing to medical intervention and, more recently, because of the possibility of genetic therapy, the elimination of some harmful mutations from the population is no longer taking place as rapidly and effectively as it did in the past.

Molecular biology has introduced in modern medicine a new way to cure diseases, namely genetic therapy, direct intervention in the genetic makeup of an individual. Gene therapy can be somatic or germ line. Germ-line genetic therapy would seek to correct a genetic defect, not only in the organs or tissues impacted, but also in the germ line, so that the person treated would not transmit the genetic impairment to the descendants. As of now, no interventions of germ-line therapy are seriously sought by scientists, physicians, or pharmaceutical companies.

The possibility of gene therapy was first anticipated in 1972 (20). The possible objectives are to correct the DNA of a defective gene or to insert a new gene that would allow the proper function of the gene or DNA to take place. In the case of a harmful gene, the objective would be to disrupt the gene that is not functioning properly.

The eminent biologist E. O. Wilson (2014) has stated, many would think somewhat hyperbolically, that the issue of how much to use genetic engineering to direct our own evolution, is the greatest moral dilemma since God stayed the hand of Abraham (21).

The first successful interventions of gene therapy concerned patients suffering from severe combined immunodeficiency (SCID), first performed in a 4-y-old girl at the National Institutes of Health in 1990 (22), soon followed by successful trials in other countries (23). Treatments were halted temporarily from 2000 to 2002 in Paris, when 2 of about 12 treated children developed a leukemia-like condition, which was indeed attributed to the gene therapy treatment. Since 2004, successful clinical trials for SCID have been performed in the United States, United Kingdom, France, Italy, and Germany (24, 25).

Gene therapy treatments are still considered experimental. Successful clinical trials have been performed in patients suffering from adrenoleukodystrophy, Parkinsons disease, chronic lymphocytic leukemia, acute lymphocytic leukemia, multiple myeloma, and hemophilia (26, 27). Initially, the prevailing gene therapy methods involved recombinant viruses, but nonviral methods (transfection molecules) have become increasingly successful. Since 2013, US pharmaceutical companies have invested more than $600 million in gene therapy (28). However, in addition to the huge economic costs, technical hurdles remain. Frequent negative effects include immune response against an extraneous object introduced into human tissues, leukemia, tumors, and other disorders provoked by vector viruses. Moreover, the genetic therapy corrections are often short lived, which calls for multiple rounds of treatment, thereby increasing costs and other handicaps. In addition, many of the most common genetic disorders are multifactorial and are thus beyond current gene therapy treatment. Examples are diabetes, high blood pressure, heart disease, arthritis, and Alzheimers disease, which at the present state of knowledge and technology are not suitable for gene therapy.

If a genetic defect is corrected in the affected cells, tissues, or organs, but not in the germ line, the ova or sperm produced by the individual will transmit the defect to the progeny. A deleterious gene that might have been reduced in frequency or eliminated from the population, owing to the death or reduced fertility of the carrier, will now persist in the population and be added to its load of hereditary diseases. A consequence of genetic therapy is that the more hereditary diseases and defects are cured today, the more of them will be there to be cured in the succeeding generations. This consequence follows not only from gene therapy but also from typical medical treatments.

The Nobel laureate geneticist H. J. Muller eloquently voiced this concern about the cure, whether through genetic therapy or traditional medical treatment, of genetic ailments. The more sick people we now cure and allow them to reproduce, the more there will be to cure in the future. The fate toward which mankind is drifting is painted by Muller in somber colors. The amount of genetically caused impairment suffered by the average individualmust by that time have grown.[P]eoples time and energywould be devoted chiefly to the effort to live carefully, to spare and to prop up their own feebleness, to soothe their inner disharmonies and, in general, to doctor themselves as effectively as possible. For everyone would be an invalid, with his own special familial twists. (ref. 29; Fig. 1).

The bionic human, on the cover of Science: an image that could represent how H. J. Muller anticipates the human condition, a few centuries hence, showing the accumulation of physical handicaps as a consequence of the medical cure of hereditary diseases. Image by Cameron Slayden and Nathalie Cary; reprinted with permission from AAAS.

It must be pointed out that the population genetic consequences of curing hereditary diseases are not as immediate (a few centuries hence) as Muller anticipates. Consider, as a first example, we look at the recessive hereditary condition of PKU. The estimated frequency of the gene is q = 0.01; the expected number of humans born with PKU is q2 = 0.0001, 1 for every 10,000 births. If all PKU individuals are cured all over the world and all of them leave as many descendants, on the average, as other humans, the frequency of the PKU allele will double after 1/q = 1/0.01 = 100 generations. If we assume 25 y per generation, we conclude that after 2,500 y, the frequency of the PKU allele will be q = 0.02, and q2 = 0.0004, so that 4 of every 10,000 persons, rather than only 1, will be born with PKU.

In the case of dominant lethal diseases, the incidence is determined by the mutation frequency of the normal to the disease allele, which is typically of the order of m = 106108, or between one in a million and one in one hundred million. Assuming the highest rate of m = 106, the incidence of the disease after 100 generations will become 1 for every 10,000 births. It would therefore seem likely that much earlier than 2,500 y, humans are likely to find ways of correcting hereditary ailments in the germ line, thereby stopping their transmission.

It must be pointed out that, although the proportion of individuals affected by any one serious hereditary infirmity is relatively small, there are many such hereditary ailments, which on the aggregate make the problem very serious. The problem becomes more serious when mental defects are taken into consideration. As pointed out above, more than 100 million people in the world suffer from mental impairments due in good part to the genetic endowment they inherited from their parents.

Human cloning may refer to therapeutic cloning, particularly the cloning of embryonic cells to obtain organs for transplantation or for treating injured nerve cells and other health purposes. Human cloning more typically refers to reproductive cloning, the use of somatic cell nuclear transfer (SCNT) to obtain eggs that could develop into adult individuals.

Human cloning has occasionally been suggested as a way to improve the genetic endowment of mankind, by cloning individuals of great achievement, for example, in sports, music, the arts, science, literature, politics, and the like, or of acknowledged virtue. These suggestions seemingly have never been taken seriously. However, some individuals have expressed a wish, however unrealistic, to be cloned, and some physicians have on occasion advertised that they were ready to carry out the cloning (30). The obstacles and drawbacks are many and insuperable, at least at the present state of knowledge.

Biologists use the term cloning with variable meanings, although all uses imply obtaining copies more or less precise of a biological entity. Three common uses refer to cloning genes, cloning cells, and cloning individuals. Cloning an individual, particularly in the case of a multicellular organism, such as a plant or an animal, is not strictly possible. The genes of an individual, the genome, can be cloned, but the individual itself cannot be cloned, as it will be made clear below.

Cloning genes or, more generally, cloning DNA segments is routinely done in many genetics and pharmaceutical laboratories throughout the world (12, 31). Technologies for cloning cells in the laboratory are seven decades old and are used for reproducing a particular type of cell, for example a skin or a liver cell, in order to investigate its characteristics.

Individual human cloning occurs naturally in the case of identical twins, when two individuals develop from a single fertilized egg. These twins are called identical, precisely because they are genetically identical to each other.

The sheep Dolly, cloned in July 1996, was the first mammal artificially cloned using an adult cell as the source of the genotype. Frogs and other amphibians were obtained by artificial cloning as early as 50 y earlier (32).

Cloning an animal by SCNT proceeds as follows. First, the genetic information in the egg of a female is removed or neutralized. Somatic (i.e., body) cells are taken from the individual selected to be cloned, and the cell nucleus (where the genetic information is stored) of one cell is transferred with a micropipette into the host oocyte. The egg, so fertilized, is stimulated to start embryonic development (33).

Can a human individual be cloned? The correct answer is, strictly speaking, no. What is cloned are the genes, not the individual; the genotype, not the phenotype. The technical obstacles are immense even for cloning a humans genotype.

Ian Wilmut, the British scientist who directed the cloning project, succeeded with Dolly only after 270 trials. The rate of success for cloning mammals has notably increased over the years without ever reaching 100%. The animals presently cloned include mice, rats, goats, sheep, cows, pigs, horses, and other mammals. The great majority of pregnancies end in spontaneous abortion (34). Moreover, as Wilmut noted, in many cases, the death of the fetus occurs close to term, with devastating economic, health, and emotional consequences in the case of humans (35).

In mammals, in general, the animals produced by cloning suffer from serious health handicaps, among others, gross obesity, early death, distorted limbs, and dysfunctional immune systems and organs, including liver and kidneys, and other mishaps. Even Dolly had to be euthanized early in 2003, after only 6 y of life, because her health was rapidly decaying, including progressive lung disease and arthritis (35, 36).

The low rate of cloning success may improve in the future. It may be that the organ and other failures of those that reach birth will be corrected by technical advances. Human cloning would still face ethical objections from a majority of concerned people, as well as opposition from diverse religions. Moreover, there remains the limiting consideration asserted earlier: it might be possible to clone a persons genes, but the individual cannot be cloned. The character, personality, and the features other than anatomical and physiological that make up the individual are not precisely determined by the genotype.

The genetic makeup of an individual is its genotype. The phenotype refers to what the individual is, which includes not only the individuals external appearance or anatomy, but also its physiology, as well as behavioral predispositions and attributes, encompassing intellectual abilities, moral values, aesthetic preferences, religious values, and, in general, all other behavioral characteristics or features, acquired by experience, imitation, learning, or in any other way throughout the individuals life, from conception to death. The phenotype results from complex networks of interactions between the genes and the environment.

A persons environmental influences begin, importantly, in the mothers womb and continue after birth, through childhood, adolescence, and the whole life. Impacting behavioral experiences are associated with family, friends, schooling, social and political life, readings, aesthetic and religious experiences, and every event in the persons life, whether conscious or not. The genotype of a person has an unlimited number, virtually infinite, of possibilities to be realized, which has been called the genotypes norm of reaction, only one of which will be the case in a particular individual (37). If an adult person is cloned, the disparate life circumstances experienced many years later would surely result in a very different individual, even if anatomically the individual would resemble the genomes donor at a similar age.

An illustration of environmental effects on the phenotype, and of interactions between the genotype and the environment, is shown in Fig. 2 (38). Three plants of the cinquefoil, Potentilla glandulosa, were collected in Californiaone on the coast at about 100 ft above sea level (Stanford), the second at about 4,600 ft (Mather), and the third in the Alpine zone of the Sierra Nevada at about 10,000 ft above sea level (Timberline). From each plant, three cuttings were obtained in each of several replicated experiments, which were planted in three experimental gardens at different altitudes, the same gardens from which the plants were collected. The division of one plant ensured that all three cuttings planted at different altitudes had the same genotype; that is, they were genetic clones from one another. (P. glandulosa, like many other plants, can be reproduced by cuttings, which are genetically identical.)

Interacting effects of the genotype and the environment on the phenotype of the cinquefoil Pontentilla glandulosa. Cuttings of plants collected at different altitudes were planted in three different experimental gardens. Plants in the same row are genetically identical because they have been grown from cuttings of a single plant; plants in the same column are genetically different but have been grown in the same experimental garden. Reprinted with permission from ref. 13.

Comparison of the plants in any row shows how a given genotype gives rise to different phenotypes in different environments. Genetically identical plants (for example, those in the bottom row) may prosper or not, even die, depending on the environmental conditions. Plants from different altitudes are known to be genetically different. Hence, comparison of the plants in any column shows that in a given environment, different genotypes result in different phenotypes. An important inference derived from this experiment is that there is no single genotype that is best in all environments.

The interaction between the genotype and the environment is similarly significant, or even more so, in the case of animals. In one experiment, two strains of rats were selected over many generations; one strain for brightness at finding their way through a maze and the other for dullness (Fig. 3; ref. 39). Selection was done in the bright strain by using the brightest rats of each generation to breed the following generation, and in the dull strain by breeding the dullest rats of every generation. After many generations of selection, the descendant bright rats made only about 120 errors running through the maze, whereas dull rats averaged 165 errors. That is a 40% difference. However, the differences between the strains disappeared when rats of both strains were raised in an unfavorable environment of severe deprivation, where both strains averaged 170 errors. The differences also nearly disappeared when the rats were raised with abundant food and other favorable conditions. In this optimal environment, the dull rats reduced their average number of errors from 165 to 120. As with the cinquefoil plants, we see (i) that a given genotype gives rise to different phenotypes in different environments and (ii) that the differences in phenotype between two genotypes change from one environment to anotherthe genotype that is best in one environment may not be best in another.

Results of an experiment with two strains of rats: one selected for brightness and the other for dullness. After many generations of selection, when raised in the same environment in which the selection was practiced (normal), bright rats made about 45 fewer errors than dull rats in the maze used for the tests. However, when the rats were raised in an impoverished (restricted) environment, bright and dull rats made the same number of errors. When raised in an abundant (stimulating) environment, the two strains performed nearly equally well. Reprinted with permission from ref. 13.

In the second half of the 20th century, as dramatic advances were taking place in genetic knowledge, as well as in the genetic technology often referred to as genetic engineering, some utopian proposals were advanced, at least as suggestions that should be explored and considered as possibilities, once the technologies had sufficiently progressed. Some proposals suggested that persons of great intellectual or artistic achievement or of great virtue be cloned. If this was accomplished in large numbers, the genetic constitution of mankind would, it was argued, considerably improve.

Such utopian proposals are grossly misguided. It should be apparent that, as stated above, it is not possible to clone a human individual. Seeking to multiply great benefactors of humankind, such as persons of great intelligence or character, we might obtain the likes of Stalin, Hitler, or Bin Laden. As the Nobel Laureate geneticist George W. Beadle asserted many years ago: Few of us would have advocated preferential multiplication of Hitlers genes. Yet who can say that in a different cultural context Hitler might not have been one of the truly great leaders of men, or that Einstein might not have been a political villain (8). There is no reason whatsoever to expect that the genomes of individuals with excellent attributes would, when cloned, produce individuals similarly endowed with virtue or intelligence. Identical genomes yield, in different environments, individuals who may be quite different. Environments cannot be reproduced, particularly several decades apart, which would be the case when the genotype of the persons selected because of their eminent achievement might be cloned.

Are there circumstances that would justify cloning a person, because he or she wants it? One might think of a couple unable to have children, or a man or woman who does not want to marry, or of two lesbian lovers who want to have a child with the genotype of one in an ovum of the other, or of other special cases that might come to mind (40). It must be, first, pointed out that the cloning technology has not yet been developed to an extent that would make possible to produce a healthy human individual by cloning. Second, and most important, the individual produced by cloning would be a very different person from the one whose genotype is cloned, as belabored above.

Ethical, social, and religious values will come into play when seeking to decide whether a person might be allowed to be cloned. Most people are likely to disapprove. Indeed, many countries have prohibited human cloning. In 2004, the issue of cloning was raised in several countries where legislatures were also considering whether research on embryonic stem cells should be supported or allowed. The Canadian Parliament on March 12, 2004 passed legislation permitting research with stem cells from embryos under specific conditions, but human cloning was banned, and the sale of sperm and payments to egg donors and surrogate mothers were prohibited. The French Parliament on July 9, 2004 adopted a new bioethics law that allows embryonic stem cell research but considers human cloning a crime against the human species. Reproductive cloning experiments would be punishable by up to 20 y in prison. Japans Cabinet Council for Science and Technology Policy voted on July 23, 2004 to adopt policy recommendations that would permit the limited cloning of human embryos for scientific research but not the cloning of individuals. On January 14, 2001, the British government amended the Human Fertilization and Embryology Act of 1990 by allowing embryo research on stem cells and allowing therapeutic cloning. The Human Fertilization and Embryology Act of 2008 explicitly prohibited reproductive cloning but allowed experimental stem cell research for treating diabetes, Parkinsons disease, and Alzheimers disease (41, 42). On February 3, 2014, the House of Commons voted to legalize a gene therapy technique known as mitochondrial replacement, or three-person in vitro fertilization, in which mitochondria from a donors egg cell contribute to a couples embryo (43). In the United States, there are currently no federal laws that ban cloning completely (42). Thirteen states (Arkansas, California, Connecticut, Iowa, Indiana, Massachusetts, Maryland, Michigan, North Dakota, New Jersey, Rhode Island, South Dakota, and Virginia) ban reproductive cloning, and three states (Arizona, Maryland, and Missouri) prohibit use of public funds for research on reproductive cloning (44).

Cloning of embryonic cells (stem cells) could have important health applications in organ transplantation, treating injured nerve cells, and otherwise. In addition to SCNT, the method discussed above for cloning individuals, another technique is available, induced pluripotent stem cells (iPSCs), although SCNT has proven to be much more effective and less costly. The objective is to obtain pluripotent stem cells that have the potential to differentiate in any of the three germ layers characteristic of humans and other animals: endoderm (lungs and interior lining of stomach and gastrointestinal tract), ectoderm (nervous systems and epidermal tissues), and mesoderm (muscle, blood, bone, and urogenital tissues). Stem cells, with more limited possibilities than pluripotent cells, can also be used for specific therapeutic purposes (45).

Stem cell therapy consists of cloning embryonic cells to obtain pluripotent or other stem cells that can be used in regenerative medicine, to treat or prevent all sorts of diseases, and for the transplantation of organs. At present, bone marrow transplantation is a widely used form of stem cell therapy; stem blood cells are used in the treatment of sickle cell anemia, a lethal disease when untreated, which is very common in places where malaria is rife because heterozygous individuals are protected against infection by Plasmodium falciparum, the agent of malignant malaria. One of the most promising applications of therapeutic cloning is the growth of organs for transplantation, using stem cells that have the genome of the organ recipient. Two major hurdles would be overcome. One is the possibility of immune rejection; the other is the availability of organs from suitable donors. Another regenerative medical application that might be anticipated is the therapeutic growth of nerve cells. There are hundreds of thousands of individuals throughout the world paralyzed from the neck down and confined for life to a wheelchair as a consequence of damage to the spinal cord below the neck, often as a consequence of a car accident or a fall, that interrupts the transmission of nerve activity from the brain to the rest of the body and vice versa. A small growth of nerve cells sufficient to heal the wound in the spinal cord would have enormous health consequences for the wounded persons and for society.

At present, the one gene therapy modification of the embryo that can be practiced is mitochondrial replacement (MR), legalized in the United Kingdom by the House of Commons on February 3, 2014 (43), as mentioned earlier. Mutations in the mitochondrial DNA of about 1 in 6,500 individuals account for a variety of severe and often fatal conditions, including blindness, muscular weakness, and heart failure (46). With MR, the embryo possesses nuclear DNA from the mother and father, as well as mtDNA from a donor female who has healthy mtDNA. However, MR remains technically challenging, with a low rate of success. One complicating issue is that mtDNA replacement is not 100% successful; disease-causing mutant mtDNA persists in the developing embryo and may account for eventual diseases due to heteroplasmy, at least in some tissues. A second issue of concern is that mtDNA disorders often appear late in life. It remains unknown whether the benefits of MR as currently practiced may persist in advanced age.

Author contributions: F.J.A. wrote the paper.

The author declares no conflict of interest.

This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sciences, In the Light of Evolution IX: Clonal Reproduction: Alternatives to Sex, held January 910, 2015, at the Arnold and Mabel Beckman Center of the National Academies of Sciences and Engineering in Irvine, CA. The complete program and video recordings of most presentations are available on the NAS website at http://www.nasonline.org/ILE_IX_Clonal_Reproduction.

This article is a PNAS Direct Submission.

See more here:

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Food Standards, Australia/New Zealand - Food from cloned animals (03/2016)

U.S. Food and Drug Administration (FDA) - Animal Cloning (11/2017)

BIO - All About Animal Cloning (09/2010)

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Are the Progeny of Cloned Animals Safe to Eat? (01/2008)

Is livestock cloning another form of genetic engineering? (01/2008)

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A clone is an organism that is descended from, and genetically identical to, a single common ancestor. Animals can be cloned by embryo splitting or nuclear transfer. Embryo splitting involves bisecting the multicellular embryo at an early stage of development to generate "twins". This type of cloning occurs naturally and has also been performed in the laboratory with a number of animal species.

Cloning can also be achieved by nuclear transfer where the genetic material from one cell is placed into a "recipient" unfertilized egg that has had its genetic material removed by a process called enucleation. The first mammals were cloned via nuclear transfer during the early 1980s, almost 30 years after the initial successful experiments with frogs . Numerous mammalian species have been cloned from cells of preimplantation embryos: namely mice, rats, rabbits, pigs, goats, sheep, cattle and even two rhesus monkeys.

Dollythe sheepwas the first animal to becloned via nuclear transfer from a cultured adult cell in 1996. Since then, adiverse range of adult tissues have been successfully cloned in a variety of species including cattle, mice, pigs, cats, rabbits, goats, and zebrafish.

The proportion of adult cell nuclei to develop into live offspring after transfer into an enucleated egg is very low. High rates of abortion have been observed at various stages of pregnancy after placement of the eggs containing the adult cell nuclei into recipient animals . Various abnormalities have been observed in cloned cows and mice after birth and this has been found to be somewhat dependent on the type of tissue that originated the nuclei used to make the clone. The reasons for the low efficiency of cloning by nuclear transfer are currently under investigation but it is thought that it may be related to insufficient nuclear reprogramming as the cloned nuclei goes from directing the production of an adult somatic cell to directing the production of a whole new embryo.

Cloning offers the opportunity to make transgenic animals from cultured cells that have been genetically engineered . The first genetically engineered or transgenic mammalian clones were sheep born in 1997 carrying the coding sequences for human clotting factor IX, which is an important therapeutic for hemophiliacs. One of these transgenic sheep, Polly, expressed this protein in her milk . Cloning may also be useful for the preservation of rare and endangered species.In human therapeutics,patients may be able to clone their own nuclei to make healthy tissue that could be used to replace diseased tissue without the risk of immunological rejection.

Making Genetically Engineered Clones (Data from Schnieke et al., 1997; Figure from De Berardino, 2001). Fetal cells in culture were transfected with a DNA sequence containing a selectable marker (neomycin resistance), the human gene for clotting factor IX, and a regulatory sequence to direct the gene to function only in the mammary gland. Following selection for neomycin resistance, nucleus from surviving cells were each transferred to an enucleated egg. Of the three transgenic clones born, one named POLLY survived and later secreted human clotting factor in her milk. Polly is the first transgenic mammalian clone.

1. Briggs,R, King,TJ: Transplantation of living nuclei from blastula cells into enucleated frogs' eggs. Proc.Natl.Acad.Sci.U.S.A 39: 455-463 (1952).

2. Meng,L, Ely,JJ, Stouffer,RL, Wolf,DP: Rhesus monkeys produced by nuclear transfer. Biol Reprod 57: 454-459 (1997).

3. Wilmut,I, Schnieke,AE, McWhir,J, Kind,AJ, Campbell,KH: Viable offspring derived from fetal and adult mammalian cells. Nature 385: 810-813 (1997).

4. Galli,C, Duchi,R, Moor,RM, Lazzari,G: Mammalian leukocytes contain all of the genetic information necessary for the development of a new individual. Cloning 1: 161-170 (1999).

5. Hill,JR, Burghardt,RC, Jones,K, Long,CR, Looney,CR, Shin,T, Spencer,TE, Thompson,JA, Winger,QA, Westhusin,ME: Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol Reprod 63: 1787-1794 (2000).

6. Kato,Y, Tani,T, Sotomaru,Y, Kurokawa,K, Kato,J, Doguchi,H, Yasue,H, Tsunoda,Y: Eight calves cloned from somatic cells of a single adult. Science 282: 2095-2098 (1998).

7. Kubota,C, Yamakuchi,H, Todoroki,J, Mizoshita,K, Tabara,N, Barber,M, Yang,X: Six cloned calves produced from adult fibroblast cells after long-term culture. Proc.Natl.Acad.Sci.U.S.A 97: 990-995 (2000).

8. Shiga,K, Fujita,T, Hirose,K, Sasae,Y, Nagai,T: Production of calves by transfer of nuclei from cultured somatic cells obtained from Japanese black bulls. Theriogenology 52: 527-535 (1999).

9. Wells,DN, Misica,PM, Tervit,HR: Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biol Reprod 60: 996-1005 (1999).

10. Zakhartchenko,V, Alberio,R, Stojkovic,M, Prelle,K, Schernthaner,W, Stojkovic,P, Wenigerkind,H, Wanke,R, Duchler,M, Steinborn,R, Mueller,M, Brem,G, Wolf,E: Adult cloning in cattle: potential of nuclei from a permanent cell line and from primary cultures. Mol.Reprod Dev. 54: 264-272 (1999).

11. Ogura,A, Inoue,K, Ogonuki,N, Noguchi,A, Takano,K, Nagano,R, Suzuki,O, Lee,J, Ishino,F, Matsuda,J: Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol Reprod 62: 1579-1584 (2000).

12. Wakayama,T, Perry,AC, Zuccotti,M, Johnson,KR, Yanagimachi,R: Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394: 369-374 (1998).

13. Wakayama,T, Yanagimachi,R: Cloning of male mice from adult tail-tip cells. Nat.Genet. 22: 127-128 (1999).

14. Polejaeva,IA, Chen,SH, Vaught,TD, Page,RL, Mullins,J, Ball,S, Dai,Y, Boone,J, Walker,S, Ayares,DL, Colman,A, Campbell,KH: Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407: 86-90 (2000).

15. Shin,T, Kraemer,D, Pryor,J, Liu,L, Rugila,J, Howe,L, Buck,S, Murphy,K, Lyons,L, Westhusin,M: A cat cloned by nuclear transplantation. Nature 415: 859 (2002).

16. Chesne,P, Adenot,PG, Viglietta,C, Baratte,M, Boulanger,L, Renard,JP: Cloned rabbits produced by nuclear transfer from adult somatic cells. Nature Biotechnology 20: 366-369 (2002).

17. Keefer,CL, Baldassarre,H, Keyston,R, Wang,B, Bhatia,B, Bilodeau,AS, Zhou,JF, Leduc,M, Downey,BR, Lazaris,A, Karatzas,CN: Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes. Biol Reprod 64: 849-856 (2001).

18. Lee,KY, Huang,HG, Ju,BS, Yang,ZG, Lin,S: Cloned zebrafish by nuclear transfer from long-term-cultured cells. Nature Biotechnology 20: 795-799 (2002).

19. Tsunoda,Y, Kato,Y: Recent progress and problems in animal cloning. Differentiation 69: 158-161 (2002).

20. Di Berardino,MA: Animal cloning--the route to new genomics in agriculture and medicine. Differentiation 68: 67-83 (2001).

21. Schnieke,AE, Kind,AJ, Ritchie,WA, Mycock,K, Scott,AR, Ritchie,M, Wilmut,I, Colman,A, Campbell,KH: Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278: 2130-2133 (1997).

22. Lanza,RP, Cibelli,JB, Diaz,F, Moraes,CT, Farin,PW, Farin,CE, Hammer.C.J., West,MD, Damiani,P: Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning 2: 79-90 (2000).

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Cloning | animalbiotech

Cloning, or somatic cell nuclear transfer (SCNT), is the technique used to produce Dolly the sheep, the first animal to be produced as a genetic copy of another adult.

In this procedure, the nucleus of an egg cell is removed and replaced by the nucleus of a cell from another adult. In Dollys case, the cell came from the mammary gland of an adult ewe. This nucleus contained that ewes DNA. After being inserted into the egg, the adult cell nucleus is reprogrammed by the host cell. The egg is artificially stimulated to divide and behave in a similar way to an embryo fertilised by sperm. After many divisions in culture, this single cell forms a blastocyst (an early stage embryo with about 100 cells) with almost identical DNA to the original donor who provided the adult cell a genetic clone.

At this stage, cloning can go one of two ways:

Reproductive cloningTo produce Dolly, the cloned blastocyst was transferred into the womb of a recipient ewe, where it developed and when born quickly became the worlds most famous lamb. When the cloning process is used in this way, to produce a living duplicate of an existing animal, it is commonly called reproductive cloning. This form of cloning has been successful in sheep, goats, cows, mice, pigs, cats, rabbits, gaur and dogs.Cloned animals

This form of cloning is unrelated to stem cell research. In most countries, it is illegal to attempt reproductive cloning in humans.

Therapeutic cloningIn therapeutic cloning, the blastocyst is not transferred to a womb. Instead, embryonic stem cells are isolated from the cloned blastocyst. These stem cells are genetically matched to the donor organism, holding promise for studying genetic disease. For example, stem cells could be generated using the nuclear transfer process described above, with the donor adult cell coming from a patient with diabetes or Alzheimers. The stem cells could be studied in the laboratory to help researchers understand what goes wrong in diseases like these.

Another long-term hope for therapeutic cloning is that it could be used to generate cells that are genetically identical to a patient. A patient transplanted with these cells would not suffer the problems associated with rejection.

To date, no human embryonic stem cell lines have been derived using therapeutic cloning, so both these possibilities remain very much in the future.

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What is cloning, and what does it have to do with stem ...

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27. GUSSONI E, SONEOKA Y, STRICKLAND CD, BUZNEY EA, KHAN MK, FLINT AF, KUNKEL LM, MULLIGAN RC. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999 Sep 23, 401(6751):390-4.

28. LEE SH, LUMELSKY N, STUDER L, AUERBACH JM, MCKAY RD. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000 Jun, 18(6):675-9.

29. WAKAYAMA T, TABAR V, RODRIGUEZ I, PERRY AC, STUDER L, MOMBAERTS P. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 2001 Apr 27, 292(5517):740-3.

30. LUMELSKY N, BLONDEL O, LAENG P, VELASCO I, RAVIN R, MCKAY R. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001 May 18, 292(5520):1389-94.

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32. NEGRIN RS, ATKINSON K, LEEMHUIS T, HANANIA E, JUTTNER C, TIERNEY K, HU WW, JOHNSTON LJ, SHIZURN JA, STOCKERL-GOLDSTEIN KE, BLUME KG, WEISSMAN IL, BOWER S, BAYNES R, DANSEY R, KARANES C, PETERS W, KLEIN J. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant 2000, 6(3):262-71.

33. FERRARI G, CUSELLA-DE ANGELIS G, COLETTA M, PAOLUCCI E, STORNAIUOLO A, COSSU G, MAVILIO F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998 Mar 06, 279(5356):1528-30.

34. PETERSEN BE, BOWEN WC, PATRENE KD, MARS WM, SULLIVAN AK, MURASE N, BOGGS SS, GREENBERGER JS, GOFF JP. Bone marrow as a potential source of hepatic oval cells. Science 1999 May 14, 284(5417):1168-70.

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Continued here:

2. Cloning: Definitions and Applications | Scientific and ...

There areplenty of great servicesthat can back up your files, but sometimes you need something a bit more bulletproof. Maybe you'remigrating your Windows installationto a new drive, or perhaps you want a complete one-to-one copy in case anything goes wrong. In those cases, your best bet is to clone your hard drive, creating an exact copy you can swap in and boot up right away.

Some backup services, likeIDriveandAcronis, have built-in disk-cloning features, supplementing to the normal file backup. We'll be using some free tools designed specifically for drive cloning in this guide, though. If you want a true backup solution with supplemental cloning features, check out one of the paid options. But for one-off clones (like if you're migrating your OS to a new drive), these tools will be all you need.

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For this process, you'll obviously need two drives: the source drive (with the data you want to clone), and thedestination drive(where you're cloning that datato). If you have a desktop computer and both drives are installed internally (or you're just cloning to a USB external drive for backup), great! You're ready to continue.

If, however, you're using a laptop with only one drive bay, you'll need an external SATA-to-USBadapter,dock, orenclosureto connect your bare drive to the computer. Once you've connected your drive, you can go through the cloning process, then disconnect it and install the drive internally.

In most cases, your destination drive will probably need to be as large as, or larger than, your source drive. If it isn't, you'll need tofree up spaceon your source drive andshrink the main partition downto fit. (You'll probably only need to do this if you're migrating from a hard drive to a smaller SSD; we have a separate guide on that process here.)

Windows users have lots of great cloning tools available, but we'll be usingMacrium Reflect Free. It's free, easy to use, and widely loved by many, so it's hard to go wrong.

To install Macrium Reflect, download the Home Use installer and start it up. It's just a tiny tool that will download the actual installer for you, based on the type of license you want. Choose the temporary folder for these filesI just put them in my Downloads folderand click the Download button.

Once it's finished, it'll automatically launch the Macrium installation wizard, which you can click right on throughthe default options should be fine for our purposes. You can safely delete all the installer files from your Downloads folder once the wizard has finished.

Open Macrium Reflect and you'll see a detailed list of the disks connected to your computer. You have two main options: you can directly clone one disk to another, or create an image of a disk. Cloning allows you to boot from the second disk, which is great for migrating from one drive to another. Imaging, on the other hand, allows you to store as many full, one-to-one copies of your source disk as the destination's space will allow, which is useful for backups.

Select the disk you want to copy (making sure to check the leftmost box if your disk has multiple partitions) and click "Clone This Disk" or "Image This Disk."

In the next window, choose your destination diskthe one that will house your newly copied data. Note that this will erase all data on the disk, so be careful which one you choose. If there's any old data on it, you may want to select it and click the "Delete Existing Partitions" button until the drive is empty.

If you're cloning to a larger drive, you'll want to click the "Cloned Partition Properties" button at the bottom of this window, and extend your main partition to fill up the entire space of the disk.

The next page will ask if you want to schedule this clone, which is useful if you want to regularly image your drive for backup purposes. I've skipped this, since I'm just doing a one-time clone. On the page after that, you can also save the backup and its schedule as an XML file for safe keeping, but I've unchecked that option for the same reasonI'm only doing this once for now.

Finally, Macrium Reflect will begin the cloning process. This can take some time depending on the size of your drive, so give it time to do its thing. If you cloned your drive, you should be able to boot from it now by selecting it in your BIOS. If you're imaging your drive, you can actually keep the second drive connected for future image backups.

If you're on a Mac, we recommendSuperDuperfor all your cloning needs. It's free, simple to use, and has been around for years. Download the app, open the DMG file, and double-click on the icon to install. (Don't drag it to your /Applications folder like you would most Mac apps; double-clicking on it should install it to your computer.)

Once installed, open SuperDuper and you'll be greeted with its incredibly simple, intuitive interface. In the first menu next to "Copy," select the source disk you want to clone. In the second menu, select the destination disk you're cloning tothis will fully erase the drive in that second menu, so make sure there isn't anything important on it! When you're ready, click the "Copy Now" button. The process will begin. (Yeah, it's that easy.)

This may take a while, but when it's done, you have two choices. If you want to replace your Mac's internal drive with the new drive (say, if you're migrating to a larger drive), you can open up your Mac and swap those now, then boot up as normal.

If you want to boot your cloned drive from USB, you can hold the Option key as your Mac starts up and select it from the boot list. Your cloned drive will be in the exact state your computer was during the cloning process, and you can continue working without skipping a beat.

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How to Clone a Hard Drive | PCMag

therapeutic cloning

nuclear transplantation of a patient's own cells to make an oocyte from which immune-compatible cells (especially stem cells) can be derived for transplant

Regarding human cloning, scientists and policymakers generally make a distinction betweenreproductive and therapeutic cloning.

making a full living copy of an organism

Reproductive cloning, the process used to create Dolly the sheep, involves implanting anembryo into a females uterus.

a general term for the research activity that creates a copy of some biological entity (a gene or organism or cell)

a genetically identical organism derived from a single cell

an undifferentiated cell whose daughter cells may differentiate into other cell types (such as blood cells)

Instead, therapeutic cloningfocuses on stem cells and how they develop.

producing new life or offspring

Regarding human cloning, scientists and policymakers generally make a distinction betweenreproductive and therapeutic cloning.

an animal organism in the early stages of growth

After receiving a careful burst ofelectricity, the egg begins to divide into an embryo as if sperm had fertilized it.

in an artificial environment outside the living organism

Kinds of CloningCloning is different from other forms of assisted reproduction, such as artificial inseminationor in vitro fertilization.

relating to the study of heredity and variation in organisms

In 1996, scientists in Scotland created Dolly, a sheep who was an identical genetic copy of her mother.

the basic structural and functional unit of all organisms

Officials and citizens around the world are discussing the uses of human cells in medical research and the prospect of reproducing people through cloning.

a disease or disorder that is inherited genetically

Some researchers use therapeutic cloning to understand genetic defects.

the act of planting or setting in the ground

If the implantation is successful, the embryo grows and is bornjust like any other baby.

a hollow muscular organ in the pelvic cavity of females

Reproductive cloning, the process used to create Dolly the sheep, involves implanting anembryo into a females uterus.

the introduction of semen into the oviduct or uterus by some means other than sexual intercourse

Kinds of CloningCloning is different from other forms of assisted reproduction, such as artificial inseminationor in vitro fertilization.

the full DNA sequence of an organism

A 1998 United Nations General Assembly declaration stated that Practiceswhich are contrary to human dignity, such as reproductive cloning of human beings, shall not bepermitted (Universal Declaration on the Human Genome and Human Rights).

making productive by adding nutrients

Kinds of CloningCloning is different from other forms of assisted reproduction, such as artificial inseminationor in vitro fertilization.

the introduction of semen into the genital tract of a female

Kinds of CloningCloning is different from other forms of assisted reproduction, such as artificial inseminationor in vitro fertilization.

an unborn vertebrate in the later stages of development

Despite thisversatility, stem cells do not themselves have the capacity to form a fetus or a newborn animal(COSEPUP, 2002).

(of illness) marked by gradual deterioration of organs and cells along with loss of function

They also use therapeutic cloning to learn how to renew cells or tissues in people who sufferfrom degenerative diseases or crippling injuries.

the male reproductive cell; the male gamete

In assisted reproduction, the sperm of a male donor is brought togetherwith the egg of a female donor, just like in natural reproduction.

the vascular structure in the uterus of most mammals providing oxygen and nutrients for and transferring wastes from the developing fetus

The outer layer of cellswhich would have grown into the placenta, the meansfor nutrients to pass to a growing fetusis discarded.

in a manner impossible to cure

Obtaining cells and tissues through therapeutic cloning gives agreat hope to a number of incurably ill patients, says Professor Eva Syklov, director of theInstitute of Experimental Medicine of the Academy of Sciences in Prague.

a person who makes a gift of money, property, etc.

In assisted reproduction, the sperm of a male donor is brought togetherwith the egg of a female donor, just like in natural reproduction.

provide with fertilizers or add nutrients to

After receiving a careful burst ofelectricity, the egg begins to divide into an embryo as if sperm had fertilized it.

thinking again about a choice previously made

In 2006, theAustralian parliament overturned a ban on therapeutic cloning, and a five-year ban in Russia isdue for reconsideration in 2007.

the act of making copies

Kinds of CloningCloning is different from other forms of assisted reproduction, such as artificial inseminationor in vitro fertilization.

the state of being capable of producing offspring

Fertility clinics routinely discard these unused embryos.

the study of heredity and variation in organisms

The applications of research, including applications inbiology, genetics and medicine, concerning the human genome, shall seek to offer relief fromsuffering and improve the health of individuals and humankind as a whole (Article 12).

fix or set securely or deeply

Reproductive cloning, the process used to create Dolly the sheep, involves implanting anembryo into a females uterus.

all of the living human inhabitants of the earth

The applications of research, including applications inbiology, genetics and medicine, concerning the human genome, shall seek to offer relief fromsuffering and improve the health of individuals and humankind as a whole (Article 12).

give to a charity or good cause

Cell Sources for CloningCurrently, surplus embryos donated by parents undergoing in vitro fertilization are used as asource for stem cells.

any substance that can be metabolized to give energy

The outer layer of cellswhich would have grown into the placenta, the meansfor nutrients to pass to a growing fetusis discarded.

having a wide variety of skills

Despite thisversatility, stem cells do not themselves have the capacity to form a fetus or a newborn animal(COSEPUP, 2002).

motivation based on ideas of right and wrong

While the same techniques are used in the initial stages ofboth processes (German National Ethics Council, 2004), they quickly differ in important ways(Committee on Science, Engineering, and Public Policy, 2002).

provide physical relief, as from pain

Supporters argue that therapeutic cloning holds great promise to alleviate human sufferingand advance human knowledge.

anything that is cast aside

Fertility clinics routinely discard these unused embryos.

having great diversity or variety

These cells are very versatile: all the specializedcells of the bodybone, blood, nerves, muscles, skindevelop from stem cells.

the principles of right and wrong for an individual or group

While the same techniques are used in the initial stages ofboth processes (German National Ethics Council, 2004), they quickly differ in important ways(Committee on Science, Engineering, and Public Policy, 2002).

part of an organism consisting of an aggregate of cells

They also use therapeutic cloning to learn how to renew cells or tissues in people who sufferfrom degenerative diseases or crippling injuries.

a copy that corresponds to an original exactly

Since that time, scientists in other parts of the world have produced genetic duplicates of such animals as a cow, a mouse, a cat, a dog, a horse, a pig, and even a ferret.

make a copy or equivalent of

Officials and citizens around the world are discussing the uses of human cells in medical research and the prospect of reproducing people through cloning.

a part of the cell responsible for growth and reproduction

Cloning, by contrast, involvestransferring the genetic material from the nucleus of one adult cell of an organism and placing itinto an egg whose genetic material has been removed.

animal reproductive body consisting of an ovum or embryo together with nutritive and protective envelopes; especially the thin-shelled reproductive body laid by e.g. female birds

In assisted reproduction, the sperm of a male donor is brought togetherwith the egg of a female donor, just like in natural reproduction.

the quality of being worthy of esteem or respect

A 1998 United Nations General Assembly declaration stated that Practiceswhich are contrary to human dignity, such as reproductive cloning of human beings, shall not bepermitted (Universal Declaration on the Human Genome and Human Rights).

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Cloning - Vocabulary List : Vocabulary.com

What is DNA Cloning?

DNA cloning is a molecular biology technique which is used for the creation of exact copies or clones of a particular gene or DNA.

DNA cloning is the process of making multiple copies of a particular segment of DNA.During this technique, the selected DNA fragment is inserted into a plasmid (the circular piece of DNA) using enzymes. Restriction enzymes and DNA ligase are used in the process.

The restriction enzymes are used to cut the DNA fragments at specific sequences and DNA ligase enzymes are used to join the nicks. The recombinant DNA thus produced is introduced into bacteria. These bacteria reproduce and produce an exact copy of the plasmid. These copies are known as clones.

Also Read:DNA Structure

DNA Cloning takes place in the following steps:

Two types of enzymes are used in this method:

The restriction enzymes cut the DNA at specific target sequences. The target gene is inserted into the cut site and is ligated by DNA ligase. This is known as a recombinant plasmid.

The recombinant plasmid is introduced into bacteria such as E.coli. The bacteria are subjected to very high temperatures which compel them to take up the DNA. This process is known as transformation. The plasmid contains an antibiotic resistance gene which helps them to survive in the presence of antibiotics. The plasmid containing bacteria are selected on a nutrient containing antibiotics. The transformed bacteria survive, while the ones without a plasmid die.

The plasmid containing the bacteria are cultured and the bacteria are provided with a chemical signal that helps them to target the protein. After protein production, the bacteria are split open to release it. The protein is purified and the target protein is isolated from other contents of the cell.

The DNA molecules produced through the cloning techniques are used for many purposes which include:

Also Read:Cloning Vectors

To know more about DNA cloning, its steps and importance, keep visiting BYJUS website or download BYJUS app for further reference.

DNA cloning is used to create a large number of copies of genes or a DNA segment.

Dolly, a female sheep, was the first mammal to be cloned from adult somatic cells by the process of nuclear transfer.

Tetra was the first rhesus macaque created by embryo splitting.

Cat, deer, ox, mule, dog, rabbit and rat are the animals that have been cloned.

Also Read:Recombinant DNA Technology

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DNA Cloning - Steps and Importance of DNA Cloning

In 1997, Ian Wilmut and Keith Campbell of the Roslin Institute shocked the scientific community and the world when they announced the birth of a successfully cloned sheep named Dolly. After Dolly was born, the cloningof humans seemed, at least in principle, achievable. The possibility of cloning humans sparked heated debate across the world about the acceptability and necessity of such a procedure. Some felt that biotechnology had gone a step too far while others welcomed such a development. Since then, several other species including, goats, pigs, mules, cows, mice, and cats, have been successfully cloned. The possibility of human cloning engages not only religious, social, cultural, and moral challenges but also legal and ethical issues. The debate on human cloning also raises questions of human and fundamental rights, particularly liberty of procreation, freedom of thought and scientific inquiry, and right to health. There are currently severaltypes of cloningcarried out by scientists that include cellular cloning, embryo cloning, and molecular cloning. Embryo cloning is further divided into, nuclear transfer, blastocyst division or twinning, and blastomere separation. The cloning technique used to clone Dolly was a type of nuclear transfer.

Human cloning technology, once optimized, will have the ability to help infertile couples who cannot produce sperm or eggs to have children that are genetically related to them. A couple could potentially decide to have a clone of the man born through his female partner or a clone of the woman providing the genetic material. A human clone would, therefore, become a single parent-child. Currently, treatment for infertility is not very successful. By some estimates, the success rates of infertility treatments, including IVF (in vitro fertilization) is less than 10%. The procedures are not only frustrating, but they are also expensive. In some instances, human cloning technology could be considered as the last best hope for having children for infertile couples.

The loss of a child is one of the worst tragedies that parents face. After such a painful ordeal, grief-stricken parents often wish they could have their perfect baby back. Human cloning technology could potentially allow parents to recreate a child or relative while seeking redress for their loss. Cells of a dying child could be taken andused later forcloning without consent from the parents. While the new child would not take away the memory, he/she would probably help take away some of the pain. The technology would allow parents to have a twin of their child, and like other twins, the new child would be a unique individual.

The freedom to decide whether or not to have an offspring is an important concept of personal liberty. People have the right to utilize human cloning technology in the same way they have a right to other reproductive related procedures and technologies such as the Vitro fertilization or contraceptives. A parentsright to beara child through cloning should, therefore, be respected. When the technology is established and becomes no less safe than natural reproduction, then human cloning should be allowed as a reproductive right. Cloning would also allow members of the LGBT community to have children related to them. In a lesbian couple, one of them could be cloned and brought to term in either of the women. In a gay couple, one of the men could be cloned, but the couple would need to find a woman to donate an egg and a surrogate mother to bring the embryo to term.

Current knowledge of bioengineering coupled with human cloning technology could help many parents have offspring free of defective genetic material that could cause disorders and deadly diseases. In a case where both parents have recessive genes for the fatal disease, they could avoid more traditional methods that could result in a child with dominant genes, which would consequently lead to the disease. The parents could use human cloning technology to have a childwithout the diseasesince the genetic makeup of the child would be the same as that of a parent who was cloned.

Human cloning technology could help children born with incurable diseases that can only be treated through a transplant, where donors with an organ match are not found. Cloning technology would allow a child to be cloned under reproductive purpose, which would allow the resulting clone to donate an organ such as a kidney or bone marrow. In that case, the older childwould be saved, and the younger clone child would also live since bone marrow regenerates, and humans can live with one kidney. The technology would allow a parent to save an existing life through a new life. Human cloning technology could also utilize the nuclear transplantation technique to produce human stem cells for therapeutic purposes. Stem cells from the umbilical cord could be cultured and allowed to develop into tissues such as bone marrow or a kidney when needed. Since the DNA of the new organ or bone marrow is matched to the patient, there would be a lower risk of organ rejection as a foreign matter by the patients body.

Human clones are sometimes called later-born twins by those receptive to the idea of human cloning. The term is justified by the fact that the cloned being would have the same genetic material as the original and would be born after the person who is cloned. The process of human cloning can be considered as taking human DNA and reversing its age back to zero. Some scientists believe that the technology would allow them to understand how to reverse DNA to any desirable age. Such knowledge would be seen as a step closer to a fountain of youth. Some people believe that human cloning technology would allow people to have some kind of immortality because their DNA would live on after they die.

Based on information gained from previous cloning experiments, cloned mammals die younger and suffer prematurely from diseases such as arthritis. Cloned animals also have a higher risk of developing genetic defects and being born deformed or with a disease. Studies on cloned mice have shown that they die prematurely from damaged livers, tumors, and pneumonia. Since human cloning technology is not tested, scientists cannot rule outbiological damageto the clone. The National Bioethics Advisory Commission report stated that it is morally unacceptable for anyone in the private or public sector, whether in a research or clinical setting, to attempt to create a child through somatic cell nuclear transfer cloning because it would pose unacceptable potential risks to the fetus or child. Human cloning technology would also put the mother at risk.

Dr. Leon Kass, chairman of the Presidents Council of Bioethics, has warned that studies on animal cloning suggest late-term fetal losses or spontaneous abortions occur at a higher rate in cloned fetuses than in natural pregnancies. In humans, a late-term fetal loss could significantly increase maternal mortality and morbidity. Cloning could also pose psychological risks to the mother due to the late spontaneous abortions, the birth of a child with severe health problems, or the birth of a stillborn baby.

One of the most satisfying and difficult things about being a human is developing a sense of self. It involves understanding our capabilities, strengths, needs, wants, and understanding how we fit into the community or the world. A crucial part of that process is learning from and then breaking away from parents and understanding how we are similar or different from our parents. Human cloning technology wouldpotentially diminishthe individuality or uniqueness of a cloned child. Even in instances where the child is cloned from someone other than their parents, it would not be very easy for them to develop a sense of self. It could also lead to the devaluation of clones when compared to a non-clone or original. Cloning would also infringe on the clones freedom, autonomy, and self-determination. Cloned children would be raised unavoidably in the shadow of the person they were cloned from.

Human cloning technology would, in return for compensation, provide offspring with specific genetic makeup. Cloning a child would also require some patented reproductive procedure and technology that could be sold. Consequently, human cloning technology would lead society to view children andpeople as objectsthat can be designed and manufactured with specific characteristics. Buyers would theoretically want to pay top dollar for a cloned embryo of a Nobel Prize winner, celebrity, or any other prominent figure in society.

Some experts have argued that societal hazards may be the least appreciated in discussions on human cloning technology. Such technology could, for example, lead to new and more effectiveforms of eugenics. In countries run by dictators, governments could engage in mass cloning of people who are deemed of proper genetic makeup. In democracies, human cloning technology could lead to free-market eugenics that could have a significant societal impact when coupled with bioengineering techniques. People could theoretically bioengineer their clones to have certain traits. When done on a mass scale, it would lead to a kind of a master race based on fashion.

In March 2005, the United Nations General assembly approved anon-binding Declarationthat called on UN member states to ban all forms of human cloning as incompatible with the protection of human life and human dignity. The Declaration concluded efforts that had begun in 2001 with a proposal from Germany and France for a convention against the reproductive cloning of humans. The US and 83 other nations supported a ban on all human cloning technology for reproductive and therapeutic or experimental purposes. The other 34 nations, including the UK, Japan, and China, voted against the ban. While 37 countries abstained from the vote, and 36 countries were absent.

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The Big Debate: Should Human Cloning Be Legalised ...

Over the past few decades molecular biologists have developed procedures to simplify and standardize cloning processes, allowing vast arrays of artificial DNA structures to be more easily assembled.

Are you familiar with all the cloning options out there?

Lets look at five different cloning methods you can use to get your construct. At the end of this article, you can find the recommended protocols for each method.

Long considered the traditional cloning method, restriction ligation cloning permits the insertion of a DNA fragment of interest into a vector through a cut and paste procedure. This inexpensive, flexible method can be broken down into a basic, two-step process.

It is important to note that restriction ligation has limitations, particularly when choosing restriction enzyme cutting site(s). Additionally, it is essential to choose buffers wisely, as not all restriction enzymes work equally well in all buffers. Moreover, even when you follow the protocol to the letter, restriction ligation isnt a flawless process, and can sometimes fail. Here are some tips to prevent this problem as much as possible.

This cloning method was commercially established in the late 1990s and has the primary advantage that one single recombination reaction moves a piece of DNA from one plasmid into another. This simplifies the process and reduces the time compared to restriction ligation cloning. While the cloning process can be completed in just 90 minutes, it is important to note that initial setup can be timely.

To perform Gateway cloning, you must first prepare your DNA fragment of interest by surrounding it with specific recombination sites (also known as Gateway recombination sites; ATT sequences). Therefore, you must first clone your DNA fragment into a donor plasmid. This can be done through traditional cloning methods or by using TOPO cloning or the Gateway BP Clonase reaction. The resulting plasmid is called an Entry Clone.

Once the DNA fragment is in an Entry Clone, it can be rapidly shuttled into any compatible GatewayDestinationvector. Thus, you can clone your gene of interest one time by restriction enzyme cloning and then use bacterial recombination to easily transfer it into a series of plasmids. This method is very useful both for transferring many DNA fragments into one type of plasmid or into many different types of plasmids.

Although specific plasmids need to be used, this method is universal for all types of DNA fragments, and has an accuracy rate of over 90%.

The Gibson Assembly method, often compared toSLIC, is the process whereby many DNA fragments are added to a construct all within a single test-tube reaction, producing clones without any scarring. You can assemble multiple parts at the same time to have flexible sequence design, and the ability to introduce promoters, terminators, and other short sequences into the assembly. Assembly is completed in under 2 hours.

Gibson Assembly does not rely on the presence of restriction sites within a particular sequence to be synthesized or cloned, so you have complete control over what is assembled. You also avoid the inclusion of unwanted additional sequences, which is often used to facilitate the cloning of multiple DNA sequences. Therefore, a greater number of DNA fragments can be joined in a single reaction with greater efficiency than conventional methods. However, beware: there is a sharp decrease in success rate when assembling more than 5 fragments at one time. Moreover, because both a polymerase and ligase are utilized during the cloning reaction, Gibson Assembly can result in sequence errors due to nucleotide mis-incorporations. As an alternative, In-Fusion cloning minimizes these errors with ligation-free technology. In-Fusion kits come as all-in-one solutions from PCR, to purification, cloning, and cells for transformation.

Golden Gate Assembly uses two Type IIS Restriction Enzymes, which cut DNA outside of the actual recognition site for the enzyme. The recognition sites are separated by at least one base pair from the sequence overhang, ensuring no scarring of the DNA sequence because the overhang sequence is not dictated by the restriction enzyme. If the recognition sequence is not palindromic, you can assemble multiple fragments at the same time and in an ordered manner. In addition, because the restriction site is altered during the reaction, digestion and ligation can happen in one tube, at the same time. Thus, this method is often considered a one pot wonder.

The Golden Gate ligation process is close to 100% efficient thanks to re-digestion mechanisms. Also, re-ligation is prevented, because cleaving outside of restriction enzymes sites removes them from the product. However, you might find that designing the right overhang sequences can be tedious, and Golden Gate Assembly is much less sequence independent than other cloning methods. Despite these limitations, Golden Gate Assembly is particularly good for constructing combinatorial libraries in which every fragment is flanked by the same two overhang sequences.

TOPO cloning uses a single enzyme, Topoisomerase I (TI) to both unwind and ligate DNA. TI is used in the natural process of replication; the opening/unwinding of DNA creates pressure further upstream, so to relieve this stress and prevent breakage TI binds to DNA, cleaves and unwinds it, then re-joins the nick just created.

TI is very efficient; you can complete a reaction in just 5 minutes at room temperature, and you dont need to use restriction enzymes or ligase. The TI is cleaved out of the scene leaving a sealed DNA fragment of interest inserted into the vector. Despite the rapid reaction time, you might spend some time with the initial preparation as specific primers are needed. In addition, the efficiency of TOPO cloning can vary depending on the polymerase used.

Cloning continues to make possible the study of the structure and function of genes and other important DNA sequences from even the most complex of genomes.

Further simplifying the cloning process are free programs like Genome Compiler that allow biologists to design DNA constructs by effortlessly simulating some of the cloning methods described in this article. Software such as Genome Compiler saves time by eliminating errors from the design, and allows users to easily order inserts or primers directly through the design software platform.

Cloning technology is consistently improving, becoming simpler and less expensive along the way. Be sure to stay up to date on these techniques to most efficiently design and synthesize your DNA.

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Cloning Methods: 5 Different Ways to Assemble

Cloning produced by humans is called artificial cloning. Since ancient times, people have cloned plants by planting stems or leaves clipped from other plants. The cloning of animals, however, was not developed until the 1900s. There are two main types of animal cloning: reproductive and therapeutic.

Reproductive cloning is the creation of offspring that are identical to an original animal. To accomplish this, scientists take DNA out of a cell from the original animal. They then take an egg from another animal and remove that eggs DNA. Next, they put the original animals DNA into the empty egg. The egg then develops into an embryo, or early form of the cloned animal. Finally, the embryo is placed within a female animal, who carries the clone until it is ready to be born.

Reproductive cloning is not easy. Cloned embryos often fail to develop. The first successfully cloned adult mammal was a sheep. The clone, named Dolly, was born in Scotland in 1996. Scientists later cloned pigs, mice, dogs, horses, and other animals. Cloning monkeys and apes has proved to be more difficult.

Some scientists have experimented with cloning human beings. Their work has been limited, however, because many people think human cloning is wrong. In 2005 the United Nations passed a declaration against human cloning.

Therapeutic cloning is the use of cloning to treat human diseases and disorders. It is still being researched. The goal is to produce healthy new cells by cloning a patients own cells. The new cells would be transplanted into the patients body, where they could replace damaged cells. Because the new cells would contain the patients own DNA, they would not be rejected by the immune system.

Scientists think that cloned cells might be used to treat Alzheimers disease, spinal cord injuries, and other serious conditions. However, many people object to the way the cloned cells would be produced. A human egg cell (from a donor) would be implanted with DNA from the patient, resulting in an embryo. This embryo would develop until special cells, called stem cells, could be taken from it. Then the embryo would be destroyed.

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