CHAPTER 1 INTRODUCTION Biotechnology (sometimes shortened to "biotech") is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose. Modern use of similar terms includes genetic engineering as well as cell- and tissue culture technologies. The concept encompasses a wide range of procedures (and history) for modifying living organisms according to human purposes — going back to domestication of animals, cultivation of plants, and "improvements" to these through breeding programs that employ artificial selection and hybridization. By comparison to biotechnology, bioengineering is generally thought of as a related field with its emphasis more on higher systems approaches (not necessarily altering or using biological materials directly) for interfacing with and utilizing living things. The United Nations Convention on Biological Diversity defines biotechnology as: "Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use." In other terms: "Application of scientific and technical advances in life science to develop commercial products" is biotechnology. Biotechnology draws on the pure biological sciences (genetics, microbiology, animal cell culture, molecular biology, biochemistry, embryology, cell biology) and in many instances is also dependent on knowledge and methods from outside the sphere of biology (chemical engineering, bioprocess engineering, information technology, biorobotics). Conversely, modern biological sciences (including even concepts such as molecular ecology) are intimately entwined and dependent on the methods developed through biotechnology and what is commonly thought of as the life sciences industry. One example of biotechnology is cloning. We have been cloning plants for centuries. Each time a leaf is excised from a violet plant and placed in soil to grow a new plant, cloning has occurred. Today, we are not only doing the physical manipulation at the visual level but also on the molecular level. In modern or molecular biotechnology, we physically select the desired characteristic at the molecular level and add it to the organism's genetic makeup. 1 What is cloning? Cloning is a complex process that lets one exactly copy the genetic, or inherited, traits of an animal (the donor). Animals can be cloned by embryo splitting or nuclear transfer. Embryo splitting involves splitting 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 species. Cloning can also be achieved by nuclear transfer where the genetic material in the nucleus from one cell is placed into a “recipient” unfertilized egg that has had its genetic material (nucleus) removed by a process called enucleation. It is then necessary to activate the egg to start dividing as if it had been fertilized. In mammals the egg must then be artificially placed into the womb of a surrogate mother where it will grow until birth. The first mammals cloned by this process were born during the mid 1980s, almost 30 years after the initial successful experiments with frogs. Numerous mammalian species have been cloned via this procedure, including mice, rats, rabbits, pigs, goats, sheep, cattle, horse, and rhesus monkeys. There are no documented cases of human cloning. 2 CHAPTER 2 BIOTECHNOLOGY IN ANIMALS 1. Biotechnology in animals a. Artificial Insemination Artificial insemination, or AI, is the process by which sperm is placed into the reproductive tract of a female for the purpose of impregnating the female by using means other than sexual intercourse or natural insemination. In humans, it is used as assisted reproductive technology, using either sperm from the woman's male partner or sperm from a sperm donor (donor sperm) in cases where the male partner produces no sperm or the woman has no male partner (i.e., single women, lesbians). In cases where donor sperm is used the woman is the gestational and genetic mother of the child produced, and the sperm donor is the genetic or biological father of the child. Artificial insemination is widely used for livestock breeding, especially for dairy cattle and pigs. 3 b. Embryo Transfer Embryo transfer refers to a step in the process of assisted reproduction in which embryos are placed into the uterus of a female with the intent to establish a pregnancy. This technique (which is often used in connection with in vitro fertilization (IVF)), may be used in humans or in animals, in which situations the goals may vary. c. Semen Presessing (parting spermatozoa X and Y) This method can make farmer controls the gender of their livestock animals. The search begins with conditioning female livestock’s reproduction organ so that the environment will be better for spermatozoa X than spermatozoa Y, or vice versa. d. In Vitro Fertilization In Vitro Fertilization is commonly referred to as IVF. IVF is the process of fertilization by manually combining an egg and sperm in a laboratory dish. When the IVF procedure is successful, the process is combined with a procedure known as embryo transfer, which is used to physically place the embryo in the uterus. e. Cloning A clone is a genetic copy of another living organism. The genetic material of a cloned offspring is drawn from a single source, rather than being a combination of sperm and egg genes. In sexual reproduction, half of the genetic material of an individual comes from a female and half from a male. Two processes can be used to obtain clones. The first process called “embryo splitting” is similar to what happens naturally when identical twins are produced. In the laboratory, an embryo is obtained by joining a sperm cell from a male donor with an egg cell from a female donor. When this embryo starts to divide into two cells, these are separated and implanted in 4 different foster mothers. For cattle, the embryo can be at the 4- or 8-cell division stage when it is separated into individual cells. This provides the potential to create 4 or 8 clones simultaneously. This method can also be used for primates. The second method is called nuclear transfer (NT) or cell nuclear replacement (CNR) and it was used to produce Dolly the sheep, the first animal cloned from a cell taken from an adult animal. In this technique, the donor nucleus containing the genetic material of a cell is introduced into an unfertilized egg cell from which the nucleus has been removed. An electrical pulse is used to fuse the donor nucleus and the egg cell together and to activate the development of the “reconstructed embryo”. The embryo is cultured a few days in the laboratory and if it develops normally, it is then implanted in a foster mother. The genetic make-up of this embryo is identical to that of the donor of the nucleus. The donor nucleus can be obtained from embryonic cells that are not fully differentiated into skin, heart, brain, etc. cells; this technique has been used for the cloning of livestock animals for about 12 years. The donor nucleus can also originate from the differentiated cells of an adult organism. Prior to the birth of Dolly in 1996, scientists thought that differentiated adult cells could not revert back and be reprogrammed to develop into a new embryo. Success rates for NT are low. f. Transgenic Animal There are various definitions for the term transgenic animal. The Federation of European Laboratory Animal Associations defines the term as an animal in which there has been a deliberate modification of its genome, the genetic makeup of an organism responsible for inherited characteristics. The nucleus of all cells in every living organism contains genes made up of DNA. These genes store information that regulates how our bodies form and function. Genes can be altered artificially, so that some characteristics of an animal are changed. For example, an embryo can have an extra, functioning gene from another source artificially introduced into it, or a gene introduced which can knock out the functioning of another particular gene in the embryo. Animals that have their DNA manipulated in this way are knows as transgenic animals. The majority of transgenic animals produced so far are mice, the animal that pioneered the technology. The first successful transgenic animal was a mouse. A few years later, it was followed by rabbits, pigs, sheep, and cattle. Why are these animals being produced? The two most common reasons are: 1. Some transgenic animals are produced for specific economic traits. For example, transgenic cattle were created to produce milk containing particular human proteins, which may help in the treatment of human emphysema. 5 2. Other transgenic animals are produced as disease models (animals genetically manipulated to exhibit disease symptoms so that effective treatment can be studied). For example, Harvard scientists made a major scientific breakthrough when they received a U.S. patent (the company DuPont holds exclusive rights to its use) for a genetically engineered mouse, called OncoMouse® or the Harvard mouse, carrying a gene that promotes the development of various human cancers. How are transgenic animals produced? Since the discovery of the molecular structure of DNA by Watson and Crick in 1953, molecular biology research has gained momentum. Molecular biology technology combines techniques and expertise from biochemistry, genetics, cell biology, developmental biology, and microbiology. Scientists can now produce transgenic animals because, since Watson and Crick’s discovery, there have been breakthroughs in: 1. 2. 3. 4. Recombinant DNA (artificially-produced DNA) Genetic cloning Analysis of gene expression (the process by which a gene gives rise to a protein) Genomic mapping The underlying principle in the production of transgenic animals is the introduction of a foreign gene or genes into an animal (the inserted genes are called transgenes). The foreign genes “must be transmitted through the germ line, so that every cell, including germ cells, of the animal contain the same modified genetic material.” (Germ cells are cells whose function is to transmit genes to an organism’s offspring.) To date, there are three basic methods of producing transgenic animals: 1. 2. 3. DNA microinjection Retrovirus-mediated gene transfer Embryonic stem cell-mediated gene transfer Gene transfer by microinjection is the predominant method used to produce transgenic farm animals. Since the insertion of DNA results in a random process, transgenic animals are mated to ensure that their offspring acquire the desired transgene. However, the success rate of producing transgenic animals individually by these methods is very low and it may be more efficient to use cloning techniques to increase their numbers. For example, gene transfer studies revealed that only 0.6% of transgenic pigs were born with a desired gene after 7,000 eggs were injected with a specific transgene. 6 1. DNA Microinjection The mouse was the first animal to undergo successful gene transfer using DNA microinjection. This method involves: a. transfer of a desired gene construct (of a single gene or a combination of genes that are recombined and then cloned) from another member of the same species or from a different species into the pronucleus of a reproductive cell b. the manipulated cell, which first must be cultured in vitro (in a lab, not in a live animal) to develop to a specific embryonic phase, is then transferred to the recipient female 2. Retrovirus-Mediated Gene Transfer A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. This method involves: a. retroviruses used as vectors to transfer genetic material into the host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic constitution b. chimeras are inbred for as many as 20 generations until homozygous (carrying the desired transgene in every cell) transgenic offspring are born The method was successfully used in 1974 when a simian virus was inserted into mice embryos, resulting in mice carrying this DNA. 3. Embryonic Stem Cell-Mediated Gene Transfer This method involves: a. isolation of totipotent stem cells (stem cells that can develop into any type of specialized cell) from embryos b. the desired gene is inserted into these cells c. cells containing the desired DNA are incorporated into the host’s embryo, resulting in a chimeric animal Unlike the other two methods, which require live transgenic offspring to test for the presence of the desired transgene, this method allows testing for transgenes at the cell stage. 7 How do transgenic animals contribute to human welfare? The benefits of these animals to human welfare can be grouped into areas: 1. 2. 3. Agriculture Medicine Industry The examples below are not intended to be complete but only to provide a sampling of the benefits. 1. Agricultural Applications a) Breeding Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate). Traditional breeding is a time-consuming, difficult task. When technology using molecular biology was developed, it became possible to develop traits in animals in a shorter time and with more precision. In addition, it offers the farmer an easy way to increase yields. b) Quality Transgenic cows exist that produce more milk or milk with less lactose or cholesterol, pigs and cattle that have more meat on them, and sheep that grow more wool. In the past, farmers used growth hormones to spur the development of animals but this technique was problematic, especially since residue of the hormones remained in the animal product. c) Disease Resistance Scientists are attempting to produce disease-resistant animals, such as influenzaresistant pigs, but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals. 2. Medical Applications a) Xenotransplantation Patients die every year for lack of a replacement heart, liver, or kidney. For example, about 5,000 organs are needed each year in the United Kingdom alone. Transgenic pigs may provide the transplant organs needed to alleviate the shortfall. Currently, xenotransplantation is hampered by a pig protein that can cause donor rejection but research is underway to remove the pig protein and replace it with a human protein. b) Nutritional Supplements and Pharmaceuticals Products such as insulin, growth hormone, and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows, sheep, or goats. Research is also underway to manufacture milk through transgenesis for treatment of 8 debilitating diseases such as phenylketonuria (PKU), hereditary emphysema, and cystic fibrosis. In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk at 2.4 grams per litre. This transgenic milk is a more nutritionally balanced product than natural bovine milk and could be given to babies or the elderly with special nutritional or digestive needs. Rosie’s milk contains the human gene alphalactalbumin. c) 3. Human Gene Therapy Human gene therapy involves adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene. The potential for treatments for the 5,000 named genetic diseases is huge and transgenic animals could play a role. For example, the A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans. Industrial Applications In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. The goats began to manufacture silk along with their milk and secrete tiny silk strands from their body by the bucketful. By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings. Toxicity-sensitive transgenic animals have been produced for chemical safety testing. Microorganisms have been engineered to produce a wide variety of proteins, which in turn can produce enzymes that can speed up industrial chemical reactions. What are the ethical concerns surrounding transgenesis? Interestingly, the creation of transgenic animals has resulted in a shift in the use of laboratory animals — from the use of higher-order species such as dogs to lower-order species such as mice — and has decreased the number of animals used in such experimentation, especially in the development of disease models. This is certainly a good turn of events since transgenic technology holds great potential in many fields, including agriculture, medicine, and industry. g. Gamete and Embryo Cryopreservation Cryopreservation—the ability to freeze and thaw with retention of viability—provides flexibility in human infertility therapy when gametes or embryos are handled in vitro because frozen tissue can be stored indefinitely in liquid nitrogen at -196°C. The freezing of human sperm is an established procedure that has resulted in the birth of thousands of progeny, as many as 30,000 per year. Not only can partner or donor sperm be frozen for therapeutic insemination 9 (TI) at a subsequent date, but sperm banking provides the assisted reproductive technologies (ART) couple with a backup option if a sample cannot be collected on demand or if sample quality is poor on the day eggs are available. In the case of donor insemination, samples are quarantined for 6 months before use, thereby minimizing the risk of infectious disease transmission. In other words, the donor is screened for sexually transmitted diseases at the time of collection, and the frozen sample is released for use 6 months later only after the donor passes rescreening. Pregnancies have been established with sperm stored at low temperatures for more than 10 years; however, it is generally recognized that fecundity using cryopreserved sperm is lower than that obtained with nonfrozen sperm with the possible exception of cryopreserved sperm delivered by intracytoplasmic sperm injection (ICSI). The cryopreservation of human ova would be advantageous to the patient with ovarian disease or any other condition that limits egg production. Moreover, freezing oocytes circumvents the ethical issues associated with embryo freezing. Unfortunately, the technology has not yet been perfected for egg freezing on a routine basis, despite the reports of successful pregnancies. In contrast, embryo cryopreservation is an established procedure that has been employed successfully for several years. Embryo banking was originally designed to provide alternatives for the ART couple with an embarrassment of riches. Because the risk of multiple pregnancy restricts the number of embryos that can be transferred during the treatment cycle, cryopreservation was begun to store surplus embryos for transfer at a later date to either the egg donor or to other women in the context of an embryo donor program. Additionally, embryo banking may be appropriate when embryos cannot be transferred during the egg pickup cycle. Another advantage to embryo freezing is that embryo thaw and transfer can be conducted in a natural or regulated cycle when the patient has not been subjected to controlled ovarian stimulation with exogenous hormones. h. Negative Effects of Animal Biotechnology 1. Synthetic genes and new gene products can evolve into toxic and/or immunogenic for human and animal 2. Uncontrolled and uncertain genetic reconstruction might cause genome to mutate and join, the presence of form abnormality by toxic or immunogenic, which is caused by the instability of genetic reconstruction DNA. 3. Virus in a group of genome causing diseases might be activated by genetic reconstruction 4. Deployment of antibiotic-immune gene on pathogen by horizontal gene transfer, made it unable to relieve infection 5. Increase the horizontal gene transfer and recombination, the main lane to cause disease 6. Genetic reconstruction DNA is made to attack genome and as the synthetic promoter that is able to cause cancer by the activation of oncogen (basic material of cancer cells) 10 CHAPTER 3 CLONING 1. History in Cloning The first theories and experiments with cloning began in the late 1880s as scientists sought to prove their theories about how the genetic material inside cells worked. The earliest experiments involved splitting the embryos of frogs and salamanders to see how the resulting animals would develop. As chromosomes became better understood, more experiments were done with cloning, resulting in the first cloned animals in 1952. a. History Discoveries about the nature of DNA in the 1940s made it possible for cloning experiments to progress. In 1944 it was discovered that genetic information for each cell was kept in the cell's DNA. When Oswald Avery found this genetic information, it gave scientists new ways to try to clone animals by using that genetic blueprint. b. Types The first cloned animals were northern leopard frogs that were cloned in 1952. Thomas J. King and Robert Briggs cloned 35 frog embryos and saw 27 tadpoles hatch. This first successful cloning taught scientists more about what cells needed to be used in the cloning process. King and Briggs believed, based on their clones, that young cells were more viable for the cloning process. Cells that were taken from adults resulted in abnormally developed tadpoles. c. Significance The next successful cloning experiments also resulted in cloned frogs. John Gurdon cloned South African frogs in 1962. His use of adult cells disproved the prior theory that only young cells could be used in the cloning process with success. From 1962 to 1965, Thomas King, Robert McKinnell and Marie Di Berardino created more frog clones from adult frog cells. 11 d. Considerations While animal cloning had been the focus of cloning experiments, the 1960s also saw other types of cloning. In 1964 F.C. Steward took an adult root cell from a carrot plant and successfully cloned the plant. Throughout the rest of the 1960s, scientists continued to clone frogs and to discover more about DNA. The first gene was discovered in 1969. e. Potential In 1977, the first cloned mice were created. Mouse cloning research continued, and new cloned mice were created in 1979. The first mammal was cloned in 1984. The cloned sheep was quickly followed in 1985 with cloned cattle embryos. A cow clone was created in 1986 and several calves in 1993. That same year, human embryos were cloned for the first time. In 1995 and 1996, sheep were cloned, including the famous Dolly. Since Dolly's creation, the cloning of mice and other small animals has continued, but human cloning research has been banned in many countries. f. Cloning Timeline 1885 August Weismann, professor of zoology and comparative anatomy at the University of Freiberg, theorized that the genetic information of a cell would diminish as the cell went through differentiation. 1888 Wilhelm Roux tested the germ plasm theory for the first time. One cell of a 2-cell frog embryo was destroyed with a hot needle; the result was a half-embryo, supporting Weismann's theory. 1894 Hans Dreisch isolated blastomeres from 2- and 4-cell sea urchin embryos and observed their development into small larvae. These experiments were regarded as refutations of the Weismann-Roux theory. 1901 Hans Spemann split a 2-cell newt embryo into two parts, resulting in the development of two complete larvae. 1902 Walter Sutton published "On the Morphology of the Chromosome Group in Brachyotola magna", hypothesizing that chromosomes carry the inheritance and that they occur in distinct pairs within a cell's nucleus. Sutton also argued that how chromosomes act when sex cells divide was the basis for the Mendelian Law of Heredity. 1902 German embryologist Hans Spemann split a 2-celled salamander embryo and each cell grew to adulthood, providing proof that early embryo cells carry 12 necessary genetic information. This finally disproved Weismann's 1885 theory that the amount of genetic information in cells decreases with each division. 1914 Hans Spermann conducted and early nuclear transfer experiment. 1928 Hans Spemann performed further, successful nuclear transfer experiments. 1938 Hans Spemann published the results of his 1928 primitive nuclear transfer experiments involving salamander embryos in the book "Embryonic Development and Induction." Spemann argued the next step for research should be the cloning organisms by extracting the nucleus of a differentiated cell and putting it into an enucleated egg. 1944 Oswald Avery found that a cell's genetic information was carried in DNA. 1950 First successful freezing of bull semen at -79°C for later insemination of cows was accomplished. 1952 First animal cloning: Robert Briggs and Thomas J. King cloned northern leopard frogs. 1953 Francis Crick and James Watson ,working at Cambridge's Cavendish Laboratory, discovered the structure of DNA. 1962 Biologist John Gurdon announced that he had cloned South African frogs using the nucleus of fully differentiated adult intestinal cells. This demonstrated that cells' genetic potential do not diminish as the cell became specialized. 1962 - 65 Robert G. McKinnell, Thomas J. King, and Marie A. Di Berardino produced swimming larvae from enucleated oocytes that had been injected with adult frog kidney carcinoma cell nuclei. 1963 Biologist J.B.S. Haldane coined the term "clone" in a speech entitled "Biological Possibilities for the Human Species of the Next Ten-Thousand Years." 1964 F.C. Steward grew a complete carrot plant from a fully differentiated carrot root cell. 1966 Marshall Niremberg, Heinrich Mathaei, and Severo Ochoa broke the genetic code, discovering what codon sequences specified each of the twenty amino acids. 1966 John B. Gurdon and V. Uehlinger grew adult frogs after injecting tadpole intestinal cell nuclei into enucleated oocytes. 13 1967 DNA ligase, the enzyme responsible for binding together strands of DNA, was isolated. 1969 James Shapiero and Johnathan Beckwith announced that they had isolated the first gene. 1970 Howard Temin and David Baltimore each independently isolated the first restriction enzyme. 1972 Paul Berg combined the DNA of two different organisms, thus creating the first recombinant DNA molecules. 1973 Stanley Cohen and Herbert Boyer created the first recombinant DNA organism using recombinant DNA techniques pioneered by Paul Berg. Also known as gene splicing, this technique that allows scientists to manipulate the DNA of an organism - the basis of genetic engineering. 1977 Karl Illmensee and Peter Hoppe created mice with only a single parent. 1978 David Rorvik published the novel In His Image: The Cloning of a Man. 1978 Baby Louise, the first child conceived through in vitro fertilization, was born. 1979 Karl Illmensee claimed to have cloned three mice. 1980 In the case Diamond v. Chakrabarty, the United States Supreme Court ruled that a "live, human made microorganism is patentable material." 1983 Kary B. Mullis developed the polymerase chain reaction (PCR) in 1983. This process allows for the rapid synthesis of designated fragments of DNA. 1983 Davor Solter and David McGrath tried to clone mice using their own version of the nuclear transfer method. 1983 The first human mother-to-mother embryo transfer was completed. 1983 - 86 Marie A. Di Berardino, Nancy H. Orr, and Robert McKinnell transplanted nuclei of adult frog erythrocytes, thus obtained pre-feeding and feeding tadpoles. 1984 Steen Willadsen cloned a sheep from embryo cells, the first verified example of mammal cloning using the process of nuclear transfer. 1985 Steen Willadsen used his cloning technique to duplicate prize cattle embryos. 14 1985 Ralph Brinster created the first transgenic livestock: pigs that produced human growth hormone. 1986 Using differentiated, one week old embryo cells, Steen Willadsen cloned a cow. 1986 Artificially inseminated surrogate mother Mary Beth Whitehead gave birth to Baby M. She tried and failed to retain custody. 1986 Neal First, Randal Prather, and Willard Eyestone used early embryo cells to clone a cow. October 1990 The National Institutes of Health officially launched the Human Genome Project to locate the 50,000 to 100,000 genes and sequence the estimated 3 billion nucleotides of the human genome. 1993 M. Sims and N.L. First reported the creation of calves by transfer of nuclei from cultured embryonic cells. 1993 Human embryos were first cloned. July 1995 Ian Wilmut and Keith Campbell used differentiated embryo cells to clone two sheep, named Megan and Morag. July 5, 1996 Dolly, the first organism ever to be cloned from adult cells, was born. February 23, 1997 Scientists at the Roslin Institute in Scotland officially announced the birth of "Dolly" March 4, 1997 President Clinton proposed a five year moratorium on federal and privately funded human cloning research. July 1997 Ian Wilmut and Keith Campbell, the scientists who created Dolly, also created Polly, a Poll Dorset lamb cloned from skin cells grown in a lab and genetically altered to contain a human gene. August 1997 President Clinton proposed legislation to ban the cloning of humans for at least 5 years. September 1997 Thousands of biologists and physicians signed a voluntary fiveyear moratorium on human cloning in the United States. December 5, 1997 Richard Seed announced that he intended to clone a human before federal laws could effectively prohibit the process. early January 1998 Nineteen European nations signed a ban on human cloning. 15 January 20, 1998 The Food and Drug Administration announced that it had authority over human cloning. July 1998 Ryuzo Yanagimachi, Toni Perry, and Teruhiko Wakayama announced that they had cloned 50 mice from adult cells since October, 1997. January 1998 Botechnology firm Perkin-Elmer Corporation announced that it would work with gene sequencing expert J. Craig Venture to privately map the human genome. 2. Types of Cloning a. Molecular Cloning Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. To amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations b. Cellular Cloning 1. Unicellular Organisms Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media. 16 A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders). According to this technique, a single-cell suspension of cells that have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies; each arising from a single and potentially clonal distinct cell. At an early growth stage when colonies consist of only a few of cells, sterile polystyrene rings (cloning rings), which have been dipped in grease are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth. 2. Cloning in Stem Cell Research Somatic cell nuclear transfer, known as SCNT, can also be used to create embryos for research or therapeutic purposes. The most likely purpose for this is to produce embryos for use in stem cell research. This process is also called "research cloning" or "therapeutic cloning." The goal is not to create cloned human beings (called "reproductive cloning"), but rather to harvest stem cells that can be used to study human development and to potentially treat disease. While a clonal human blastocyst has been created, stem cell lines are yet to be isolated from a clonal source. Therapeutic cloning is achieved by creating embryonic stem cells in the hopes of treating diseases such as diabetes and Alzheimer’s. The process begins by taking out the nucleus that contains the DNA from an egg and putting it in a nucleus from an adult. In the case of someone with Alzheimer’s disease, the nucleus from a skin cell of that patient is placed into an empty egg. The reprogrammed cell begins to develop into an embryo because the egg reacts with the transferred nucleus. The embryo will become genetically identical to the patient. The embryo will then form a blastocyst which has the potential to form/become any cell in the body. The reason why SCNT is used for cloning is because somatic cells can be easily acquired and cultured in the lab. This process can either add or delete specific genomes of farm animals. A key point to remember is that cloning is achieved when the oocyte maintains its normal functions and instead of using sperm and egg genomes to replicate, the oocyte is inserted into the donor’s somatic cell nucleus. The oocyte will react on the somatic cell nucleus, the same way it would on sperm cells. In SCNT, not all of the donor cell's genetic information is transferred, as the donor cell's mitochondria that contain their own mitochondrial DNA are left 17 behind. The resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. As a consequence, clones such as Dolly that are born from SCNT are not perfect copies of the donor of the nucleus. 3. Techniques of Cloning a. Somatic Cell Nuclear Transfer Somatic cell nuclear transfer, (SCNT) uses a different approach than artificial embryo twinning, but it produces the same result: an exact clone, or genetic copy, of an individual. This was the method used to create Dolly the Sheep. What does SCNT mean? Somatic cell: A somatic cell is any cell in the body other than the two types of reproductive cells, sperm and egg. Sperm and egg are also called germ cells. In mammals, every somatic cell has two complete sets of chromosomes, whereas the germ cells only have one complete set. Nuclear: The nucleus is like the cell's brain. It's an enclosed compartment that contains all the information that cells need to form an organism. This information comes in the form of DNA. It's the differences in our DNA that make each of us unique. Transfer: Moving an object from one place to another. To make Dolly, researchers isolated a somatic cell from an adult female sheep. Next, they transferred the nucleus from that cell to an egg cell from which the nucleus had been removed. After a couple of chemical tweaks, the egg cell, with its new nucleus, was behaving just like a freshly fertilized zygote. It developed into an embryo, which was implanted into a surrogate mother and carried to term. The lamb, Dolly, was an exact genetic replica of the adult female sheep that donated the somatic cell nucleus to the egg. She was the first-ever mammal to be cloned from an adult somatic cell. 18 How does SCNT differ from the natural way of making an embryo? The fertilization of an egg by a sperm and the SCNT cloning method both result in the same thing: a dividing ball of cells, called an embryo. So what exactly is the difference between these methods? An embryo is composed of cells that contain two complete sets of chromosomes. The difference between fertilization and SCNT lies in where those two sets originated. In fertilization, the sperm and egg both contain one set of chromosomes. When the sperm and egg join, the resulting zygote ends up with two sets - one from the father (sperm) and one from the mother (egg). In SCNT, the egg cell's single set of chromosomes is removed. It is replaced by the nucleus from a somatic cell, which already contains two complete sets of chromosomes. Therefore, in the resulting embryo, both sets of chromosomes come from the somatic cell. 19 b. Honolulu technique In July of 1998, a team of scientists at the University of Hawaii announced that they had produced three generations of genetically identical cloned mice. The technique is accredited to Teruhiko Wakayama and Ryuzo Yanagimachi of the University of Hawaii. Mice had long been held to be one of the most difficult mammals to clone due to the fact that almost immediately after a mouse egg is fertilized, it begins dividing. Sheep were used in the Roslin technique because their eggs wait several hours before dividing, possibly giving the egg time to reprogram its new nucleus. Even without this luxury, Wakayama and Yanagimachi were able to clone with a much higher success rate (three clones out of every one-hundred attempts) than Ian Wilmut (one in 277). Wakayama approached the problem of synchronizing cell cycles differently than Wilmut. Wilmut used udder cells, which had to be forced into the G0 stage. Wakayama initially used three types of cells, Sertoli cells, brain cells, and cumulus cells. Sertoli and brain cells both remain in the G0 state naturally and cumulus cells are almost always in either the G0 or G1 state. Unfertilized mouse egg cells were used as the recipients of the donor nuclei. After being enucleated, the egg cells had donor nuclei inserted into them. The donor nuclei were taken from cells within minutes of the cell’s extraction from a mouse. Unlike the process used to create Dolly, no in vitro, or outside of an animal, culturing was done on the cells. After one hour, the cells had accepted the new nucleus. After an additional five hours, the egg cell was then placed in a chemical culture to jumpstart the cell’s growth, just as fertilization does in nature. In the culture was a substance (cytochalasin B) which stopped the formation of a polar body, a second cell which normally forms before fertilization. The polar body would take half of the genes of the cell, preparing the other cell to receive genes from sperm. After being jumpstarted, the cells develop into embryos. These embryos can then be transplanted into surrogate mothers and carried to term. The most successful of the cells for the process were cumulus cells, so research was concentrated on cells of that type. After proving that the technique was viable, Wakayama also made clones of clones and allowed the original clones to give birth normally to prove that they had full reproductive functions. At the time he released his results, Wakayama had created fifty clones. 20 This new technique allows for further research into exactly how an egg reprograms a nucleus, since the cell functions and genomes of mice are some of the best understood. Mice also reproduce within months, much more rapidly than sheep. This aids in researching long term results. 21 c. Roslin technique The cloning of Dolly has been the most important event in cloning history. Not only did it spark public interest in the subject, but it also proved that the cloning of adult animals could be accomplished. Previously, it was not known if an adult nucleus was still able to produce a completely new animal. Genetic damage and the simple deactivation of genes in cells were both considered possibly irreversible. The realization that this was not the case came after the discovery by Ian Wilmut and Keith Cambell of a method with which to synchronize the cell cycles of the donor cell and the egg cell. Without synchronized cell cycles, the nucleus would not be in the correct state for the embryo to accept it. Somehow the donor cell had to be forced into the Gap Zero, or G0 cell stage, or the dormant cell stage. First, a cell (the donor cell) was selected from the udder cells of a Finn Dorset sheep to provide the genetic information for the clone. For this experiment, the researchers allowed the cell to divide and form a culture in vitro, or outside of an animal. This produced multiple copies of the same nucleus. This step only becomes useful when the DNA is altered, such as in the case of Polly, because then the changes can be studied to make sure that they have taken effect. A donor cell was taken from the culture and then starved in a mixture which had only enough nutrients to keep the cell alive. This caused the cell to begin shutting down all active genes and enter the G0 stage. The egg cell of a Blackface ewe was then enucleated and placed next to the donor cell. One to eight hours after the removal of the egg cell, an electric pulse was used to fuse the two cells together and, at the same time, activate the development of an embryo. This technique for mimicking the activation provided by sperm is not completely correct, since only a few electrically activated cells survive long enough to produce an embryo. If the embryo survives, it is allowed to grow for about six days, incubating in a sheep's oviduct. It has been found that cells placed in oviducts early in their development are much more likely to survive than those incubated in the lab. Finally, the embryo is placed into the uterus of a surrogate mother ewe. That ewe then carries the clone until it is ready to give birth. Assuming nothing goes wrong, an exact copy of the donor animal is born. This newborn sheep has all of the same characteristics of a normal newborn sheep. It has yet to be seen if any adverse effects, such as a higher risk of cancer or other genetic diseases that occur with the gradual damage to DNA over time, are present in Dolly or other animals cloned with this method. 22 4. The Benefits of Cloning a. Cloning for medical purposes Of all the reasons, cloning for medical purposes has the most potential to benefit large numbers of people. 1. Cloning animal models of disease Much of what researchers learn about human disease comes from studying animal models such as mice. Often, animal models are genetically engineered to carry disease-causing mutations in their genes. Creating these transgenic animals is a time-intensive process that 23 requires trial-and-error and several generations of breeding. Cloning technologies might reduce the time needed to make a transgenic animal model, and the result would be a population of genetically identical animals for study. 2. Cloning stem cells for research Stem cells are the body's building blocks, responsible for developing, maintaining and repairing the body throughout life. As a result, they might be used to repair damaged or diseased organs and tissues. Researchers are currently looking toward cloning as a way to create genetically defined human stem cells for research and medical purposes. 3. "Pharming" for drug production Farm animals such as cows, sheep and goats are currently being genetically engineered to produce drugs or proteins that are useful in medicine. Just like creating animal models of disease, cloning might be a faster way to produce large herds of genetically engineered animals. b. Cloning for agriculture purposes Another benefit from modern cloning is in agriculture. Farmers and ranchers can now have their strongest crops and animals twinned so that they are less likely to contract diseases. c. Reviving Endangered or Extinct Species Just like what had been pictured in ‘Jurassic Park’ movie. In this feature film, scientists use DNA preserved for tens of millions of years to clone dinosaurs. They find trouble, however, when they realize that the cloned creatures are smarter and fiercer than expected. Could we really clone dinosaurs? In theory? Yes. What would you need to do this? A well-preserved source of DNA from the extinct dinosaur, and A closely related species, currently living, that could serve as a surrogate mother In reality? Probably not. It's not likely that dinosaur DNA could survive undamaged for such a long time. However, scientists have tried to clone species that became extinct more recently, using DNA from wellpreserved tissue samples. d. Reproducing a Deceased Pet If we really wanted to, and if there is enough money, cloning our beloved family cat is not a dream. At least one biotechnology company in the United States offers cat cloning services for the privileged and bereaved, and they are now working to clone dogs. 24 e. Cloning Humans? To clone or not to clone: that is the question. The prospect of cloning humans is highly controversial and raises a number of ethical, legal and social challenges that need to be considered. Why would anyone want to clone humans? Some reasons include: - To help infertile couples have children To replace a deceased child 5. Pros and cons in cloning When Dolly, the first cloned sheep came in the news, cloning interested the masses. Not only researchers but even common people became interested in knowing about how cloning is done and what pros and cons it has. Everyone became more curious about how cloning could benefit the common man. Most of us want to know the pros and cons of cloning, its advantages and its potential risks to mankind. Let us understand them. a. Pros of Cloning Cloning finds applications in genetic fingerprinting, amplification of DNA and alteration of the genetic makeup of organisms. It can be used to bring about desired changes in the genetic makeup of individuals thereby introducing psitive traits in them, as also for elimination of negative traits. Cloning can also be applied to plants to remove or alter defective genes, thereby making them resistant to diseases. Cloning may find applications in development of human organs, thus making human life safer. Here we look at some of the potential advantages of cloning. 1. Organ Replacement: If the vital organs of the human body can be cloned, they can serve as backup systems for human beings. Cloning body parts can serve as a lifesaver. When a body organ such as a kidney or heart fails to function, it may be possible to replace it with the cloned body organ. 2. Substitute for Natural Reproduction: Cloning in human beings can prove to be a solution to infertility. Cloning can serve as an option for producing children. With cloning, it would be possible to produce certain desired traits in human beings. We might be able to produce children with certain qualities. Wouldn't that be close to creating a man-made being?! 3. Help in Genetic Research: Cloning technologies can prove helpful to researchers in genetics. They might be able to understand the composition of genes and the effects of genetic constituents on human traits, in a better manner. They will be able to alter 25 genetic constituents in cloned human beings, thus simplifying their analysis of genes. Cloning may also help us combat a wide range of genetic diseases. 4. Obtain Specific Traits in Organisms: Cloning can make it possible for us to obtain customized organisms and harness them for the benefit of society. Cloning can serve as the best means to replicate animals that can be used for research purposes. Cloning can enable the genetic alteration of plants and animals. If positive changes can be brought about in living beings with the help of cloning, it will indeed be a boon to mankind. b. Cons of Cloning Like every coin has two sides, cloning has its flip side too. Though cloning may work wonders in genetics, it has potential disadvantages. Cloning, as you know, is copying or replicating biological traits in organisms. Thus it might reduce the diversity in nature. Imagine multiple living entities like one another! Another con of cloning is that it is not clear whether we will be able to bring all the potential uses of cloning into reality. Plus, there's a big question of whether the common man will afford harnessing cloning technologies to his benefit. Here we look at the potential disadvantages of cloning. 1. Detrimental to Genetic Diversity: Cloning creates identical genes. It is a process of replicating a genetic constitution, thus hampering the diversity in genes. While lessening the diversity in genes, we weaken our ability of adaptation. Cloning is also detrimental to the beauty that lies in diversity. 2. Inivitation to Malpractices: While cloning allows man to tamper with genetics in human beings, it also makes deliberate reproduction of undesirable traits, a probability. Cloning of body organs might invite malpractices in society. 3. Will this Technology Reach the Common Man?: In cloning human organs and using them for transplant, or in cloning human beings themselves, technical and economic barriers will have to be considered. Will cloned organs be cost-effective? Will cloning techniques really reach the common man? 4. Man, a Man-made Being?: Moreover, cloning will put human and animal rights at stake. Will cloning fit into our ethical and moral principles? Cloning will make man just another man-made being. Won't it devalue mankind? Won't it demean the value of human life? 6. Cloning in Livestock Production Why use cloning in livestock production? Researchers around the world are investigating the potential for using cloned animals in livestock production. Cloning allows breeders to take animals with desirable traits and 26 successfully have these new traits reproduced in the offspring. Selective breeding using traditional practices does not always result in offspring with the desired traits. Cloning could be used for a dairy cow that produces milk with an unusually high milk protein content (important in cheese manufacture) or an unusually low saturated fat content (potential human health benefits), for example. Cloning could also be used for a sheep identified as superior for a particular type of wool. Researchers have also suggested that cloning could be used to preserve a species nearing extinction or to enhance livestock resistance to illnesses such as foot-and-mouth disease. The ethical and moral implications of using cloning for livestock production are also being considered. Concerns have been raised about the low success rate of the NT cloning technique and that cloning could reduce genetic diversity. Why clone? The main use of clones is to produce breeding stock, not food. Clones allow farmers to upgrade the overall quality of their herds by providing more copies of the best animals in the herd. These animals are then used for conventional breeding, and the sexually reproduced offspring become the food-producing animals. Just as farmers wouldn’t use their best conventionally bred breeding animals as sources of food, they are equally unlikely to do so for clones. Some examples of desirable characteristics in livestock that breeders might want in their herds include the following: a. Disease resistance: Sick animals are expensive for farmers. Veterinary bills add up, and unhealthy animals don’t produce as much meat or milk. A herd that is resistant to disease is extremely valuable because it doesn’t lose any production time to illness, and doesn’t cost the farmer extra money for veterinary treatment. b. Suitability to climate: Different types of livestock grow well in different climates. Some of this is natural and some results from selective breeding. For instance, Brahma cattle can cope with the heat and humidity of weather in the southwestern United States, but they often do not produce very high grades of meat. Cloning could allow breeders to select those cattle that can produce high quality meat or milk and thrive in extreme climates and use them to breed more cattle to be used for food production. Similarly, pork production has traditionally been centered in the eastern United States, but is moving to different regions of the United States (e.g., Utah). Cloning could allow breeders to select those pigs that 27 naturally do well in the new climate, and use them to breed more pigs to be used for food production. c. Quality body type: Farmers naturally want an animal whose body is well suited to its production function. For example, a dairy cow should have a large, well-attached udder so that she can produce lots of milk. She should also be able to carry and deliver calves easily. For animals that produce meat, farmers breed for strong, heavymuscled, quick-maturing animals that will yield high quality meat in the shortest time possible. The most desirable bulls produce offspring that are relatively small at birth (so that they are easier for the female to carry and deliver), but that grow rapidly and are healthy after birth. d. Fertility: Quality dairy cows should be very fertile, because a cow that doesn’t get pregnant and bear calves won’t produce milk. Male fertility is just important as that of the female. The more sperm he can produce, the more females a bull can inseminate, and the more animals will be born. Beef cattle or other meat-producing animals such as pigs need to have high fertility rates in order to replace animals that are sent to slaughter. Cloning allows farmers and breeders to clone those animals with high fertility rates so that they could bear offspring that would also tend to be very fertile. e. Market preference: Farmers or ranchers may also want to breed livestock to meet the changing tastes of consumers. The traits the producers are looking for include leanness, tenderness, color, and size of various cuts. Preferences also vary by culture, and cloning may help tailor products to the preferences of various international markets and ethnic groups. How does cloning help get these characteristics into the herd more quickly? As we’ve previously said, cloning allows the breeder to increase the number of breeding animals available to make the actual food production animals. So, if a producer wanted to introduce disease resistance into a herd rapidly, cloning could be used to produce a number of breeding animals that carry the gene for disease resistance, rather than just one. Likewise, if a breeder wants to pass on the genes of a female animal, cloning could result in multiples of that female to breed, rather than just one. 28 Is it safe to eat food from clones? It’s important to remember that the purpose of clones is for breeding, not eating. Dairy, beef, or pork clones will make up a tiny fraction of the total number of food producing animals in the United States. Instead, their offspring will be the animals actually producing meat or milk for the food supply. Dairy clones will produce milk after they give birth, and the dairy farmers will want to be able to drink that milk or put it in the food supply. Once clones used for breeding meatproducing animals can no longer reproduce, their breeders will also want to be able to put them into the food supply. In order to determine whether there would be any risk involved in eating meat or milk from clones or their offspring, in 1999 FDA asked the National Academy of Sciences (NAS) to identify science-based concerns associated with animal biotechnology, including cloning. The NAS gathered an independent group of top, peer-selected scientists from across the country to conduct this study. The scientists delivered their report in the fall of 2002. That report stated that theoretically there were no concerns for the safety of meat or milk from clones. On the other hand, the report expressed a low level of concern due to a lack of information on the clones at that time, and not for any specific scientific reasons. The report also stated that the meat and milk from the offspring of clones posed no unique food safety concerns. Meanwhile, FDA itself began the most comprehensive examination of the health of livestock clones that has been conducted. The evaluation has taken more than four years. This examination formed the basis of a Draft Risk Assessment to determine whether cloning posed a risk to animal health or to humans eating food from clones or their offspring. FDA conducted a thorough search of the scientific literature on clones, and identified hundreds of peer-reviewed scientific journal articles, which it then reviewed. They were also able to obtain health records and blood samples from almost all of the cattle clones that have been produced in the United States and data from clones produced in other countries. FDA compared these health records, and the independently analyzed blood results with similar samples from conventional animals of the same age and breed that were raised on the same farms. After reviewing all this information, FDA found that it could not tell a healthy clone from a healthy conventionally bred animal. All of the blood values, overall health records, and behaviors were in the same range for clones and conventional animals of the same breed raised on the same farms. FDA also saw that milk from dairy clones does not differ significantly in composition from milk from conventionally bred animals. 29 In the Draft Risk Assessment, FDA concluded that meat and milk from cattle, swine, and goat clones would be as safe as food we eat from those species now. It did not have enough information to make a decision on the safety of food from sheep clones. For another study similar to the one conducted on cow clones, the Agency also evaluated the health of offspring sexually derived from swine clones, as well as the composition of their meat. After reviewing this very large data set, the Agency concluded that all of the blood values, overall health records, and meat composition profiles of the progeny of clones were in the same range as for very closely genetically related conventionally bred swine. Based on these results, other studies from scientific journals, and our understanding of the biological processes involved in cloning, the Agency agreed with NAS that food from the sexually reproduced offspring of clones is as safe as food that we eat every day. These offspring animals will produce almost all of the food from the overall cloning/breeding process. 7. Cloned Animals 1. Carp An Asian carp was cloned successfully in 1963; ten years later, scientist Tong Dizhou also cloned a European crucian carp. 30 2. Dolly The Sheep Dolly saw the light of day in 1996. She lived until the age of six. The first cloned mammal, Dolly is considered to be a great success. Later, several hundred other Dollies were cloned. 3. Cumulina The Mouse 31 Cloned in Hawa’ii in 2000, Cumulina was the first successful mouse clone. She lived until the ripe old age of two years and seven months, a victory for her researchers. 4. Noto and Kaga (Cows) These cows were cloned in 1998 and duplicated several thousand times. Made in Japan, the cows pave the way for other clones engineered to produce better meat and milk. 5. Mira The Goat Also cloned in 1998, Mira and her sisters came from a US lab as predecessors for livestock engineered to contain pharmaceutical products beneficial for humans. 32 6. A Family of Pigs : Millie, Alexis, Christa, Dotcom, and Carrel Labs intend to modify pigs so that they can grow cells and organs that humans can use. Millie and her sisters (if you can call them that) were cloned in 2000 by a US-based company. 33 7. Ombretta The Mouflon The successful cloning of this endangered animal (2000) exemplifies how cloning can rescue a species from the brink of extinction. 34 8. Tetra the Rhesus Monkey The lab monkey world received its first clone in 2000. US-based Tetra is the first in a series of cloned monkeys that scientists could use as test subjects to learn more about diseases like diabetes. 35 9. Noah The Gaur A gaur is an Asian wild ox whose numbers are dwindling. Cloned in 2001, Noah only lived for two days before dying of dysentery. 36 10. Rabbit Cloned in 2001, a white rabbit like the one featured above–and its 30 clones–wasn’t given a cute name. 11. Copy Cat (CC) 37 This cat, cloned in 2001, was the starting gun for a pet-cloning process that may eventually become an industry. 12. Ralph The Rat Cloned in 2002, Ralph eventually came out of the womb 15 separate times (his clones, that is). Though rats like Ralph may eventually be used in labs, cross your fingers that his ilk won’t find their way into New York sewers. 13. Idaho Gem Mules are sterile–unless you clone them, as proven by Idaho Gem, the pride of a 2003 American research team. 38 14. Prometea The Horse An Italian team produced Prometea in 2003. They hoped to produce more Italian stallions, but their attempts failed. Prometea birthed her own in 2008. Racehorses could come in the future. 15. Diteaux The African Wildcat Although African wildcats aren’t endangered, US scientists cloned one in 2003 as a sort of template for cloning other, more vulnerable animals. 39 16. Dewey The Deer This white tail, cloned at Texas A&M University in 2003, is one of those clones lacking a solid premise. His ilk are some of the most abundant game in North America; still, scientists say clones could be used to research deer genes and produce better deer stock for hunters. As importantly, they managed to clone a deer before anyone else could do it. 17. Libby and Lilly, Ferrets 40 These ferrets, cloned in 2004, almost beg another “why the heck did you do that?” It turns out that ferrets are very useful for studying human respiratory diseases, and some types are endangered. 18. Buffalo This cloned Murrah buffalo from India could eventually become a high-volume milk source. 41 19. Snuppy The Dog South Korean scientists accomplished the notoriously challenging task of cloning a dog in 2005. Snuppy’s predecessors could be used to study human diseases. 20. Wolves : Snuwolff and Snuwolffy Seoul National University (SNU) hit the canine cloning jackpot again with these two gray wolves as precursors for eventual conservation projects in 2005. 42 8. Use Of Cloned Animals 1. The meat from cloned animals can be used as a source of food supply 2. To produce superior animals 3. To protect the endangered animals from extinction 43 CHAPTER 4 CONCLUSION AND ADVICE CONCLUSION 1. Cloning is a reproduction technology which enables to produce heir without going through the impregnation process of sperm and ovum cell as in common reproduction. 2. There are three different techniques of cloning: nucleus transfer, roslin technique, and Honolulu technique. 3. Cloning is one of the proves of the science development, and it can be used to protect animals from extinction, to develop and to increase the amount of superior breed. 4. Aside from its usefulness, cloning also has some negative effect to the animal being cloned itself, human health, the environment, and also the social-economics. ADVICE This technique should be used optimally for the development of science and to bring advantages for human. 44 Internet Resources http://animalscience.ucdavis.edu/animalbiotech/Outreach/Livestock_cloning.pdf http://www.fda.gov/AnimalVeterinary/NewsEvents/FDAVeterinarianNewsletter/ucm1081 31.htm http://learn.genetics.utah.edu/content/tech/cloning/whyclone/ http://en.wikipedia.org/wiki/ http://chestofbooks.com/animals/horses/Health-Disease-Treatment-4/ArtificialInsemination.html http://www.buzzle.com/articles/pros-and-cons-of-cloning.html http://www.actionbioscience.org/biotech/margawati.html http://www.glowm.com/index.html?p=glowm.cml/section_view&articleid=365 http://robby.nstemp.com/shopping_page.html 45