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Concepts of biotechnology Term paper animals Topic – transgenic

Submitted by : Submitted to :
Isha nayyar Harsh Btb – a Roll no.19 1040070134 Mr.

INTRODUCTION
Transgenic animals are those whose genetic material has been altered using genetic engineering techniques. These techniques are generally known as recombinant DNA technology. In other words , 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. With this technology, DNA molecules from different sources are combined into one molecule to create a new set of genes. This DNA is then transferred into an organism, giving it modified or novel traits • • • The new gene is inherited by offspring in the same way as the organism's own genes. The transgenic condition is achieved by injecting the foreign gene into the fertilized egg or into embryonic cells. The injected gene becomes part of the host cell's deoxyribonucleic acid (DNA) within the chromosome and is then inherited by all the cells produced during embryonic development. It is thus present in all the cells of the resulting adult organism and is inherited by all its descendants. This technique can be used to generate animals or plants that express genes conferring, for example, improved meat yield or resistance to specific diseases. It can also be used to create organisms that function as biological factories manufacturing hormones and other biological compounds used to treat human

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diseases. For example, bacteria can produce the hormone insulin, which is used to treat diabetes. Such uses of transgenic organisms are widely referred to as genetic engineering.

HISTORICAL DEVELOPMENT

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Prior to the development of molecular genetics, the only way of studying the regulation and function of mammalian genes was through the observation of inherited characteristics or spontaneous mutations. Long before Mendel and any molecular genetic knowledge, selective breeding was a common practice among farmers for the enhancement of chosen traits, e.g., increased milk production. During the 1970s, the first chimeric mice were produced. The cells of two different embryos of different strains were combined together at an early stage of development (eight cells) to form a single embryo that subsequently developed into a chimeric adult, exhibiting characteristics of each strain. The mutual contributions of developmental biology and genetic engineering permitted rapid development of the techniques for the creation of transgenic animals. DNA microinjection, the first technique to prove successful in mammals, was first applied to mice (Gordon and Ruddle, 1981) and then to various other species such as rats, rabbits, sheep, pigs, birds, and fish. Two other main techniques were then developed: those of retrovirus-mediated transgenesis(Jaenisch , 1976) and embryonic stem (ES) cell-mediated gene transfer (Gossler et al., 1986). Since 1981, when the term transgenic was first used by J.W. Gordon and F.H. Ruddle (1981), there has been rapid development in the use of genetically engineered animals as investigators have found an increasing number of applications for the technology.

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HOW ARE THEY CREATED ?
Materials required:
• Vectors generally used are

1. Fish vector (a plasmid) e.g - pRSV 2. P element vector (derived from drosophila and is a bacterial plasmid vector) e.g
.-pUC8. 3. BPV vectors (derived from bovine papilloma virus”transforming region” +pBR32) 4. Mammalian virus vector :SV40 5. Retrovirus Vectors(derived from pBR32+retroviruses sequences) • Promoters : a promoter sequence is the site to which RNA polymerase first binds during the initiation of transcription;the affinity of RNA Polymerase to promoter sequence in several order of magnitudes higher than that for other DNA sequences. The presence of promoter sequence is very essential for the transcription of DNA ;the promoter must be located at the end which has initiation codon AUG .e.g –RSV(Rous sarcoma virys) ,beta lactoglobulin promoter. Reporter or marker gene –produces a phenotype which is either easily or specifically detected or that allow a differential multiplication of the cells ,the former is called scorable vector while the latter is called selectable markers.e.g. of scorable are CAT gene from E.coli transposan Tn9.

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The above mentioned , are the essential elements for the creation of transgenic animals. Process :
Firstly a suitable vector is chosen and the required gene is inserted into it i.e. recombinant DNA is produced. Then,this recombinant DNA will be introduced in the host cells (cultured cells) . selection of transformed cells and identification of the cell containing the gene (using reporter or marker gene).multiplication of the introduced gene in host. Then it is transferred into an organism and that organism is called transgenic organism .

Techniques used:

1. DNA MICROINJECTION - Direct microinjection of a chosen gene construct from another member of the same species or from a different species, into the pronucleus of a fertilized ovum. 2. EMBRYONIC STEM CELL MEDIATED GENE - Insertion of desired DNA sequence into an in vitro culture of embryonic stem (ES) cells. Stem cells can give rise to a complete organism. The cells are then incorporated into an embryo at the blastocyst stage of development. 3.RETROVIRUS MEDIATED GENE TRANSFER - Retroviruses are used as vectors which infects the host cells and the retrovirus integrate into the germ cells to work.

DNA MICROINJECTION :
Method involves: • • • Microinjection refers to the process of using a very fine needle to insert substances at a microscopic or borderline macroscopic level into a single living cell. It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or the nuclear envelope. The desired contents are then injected into the desired sub-cellular compartment and the needle is removed.

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Microinjection is normally performed under a specialized optical microscope setup called a micromanipulator. The process is frequently used as a vector in genetic engineering and transgenetics to insert genetic material into a single cell. Microinjection can also be used in the cloning of organisms, and in the study of cell biology and viruses. This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum. It is one of the first methods that proved to be effective in mammals . The introduced DNA may lead to the over- or under-expression of certain genes or to the expression of genes entirely new to the animal species. The insertion of DNA is, however, a random process, and there is a high probability that the introduced gene will not insert itself into a site on the host DNA that will permit its expression. The manipulated fertilized ovum is transferred into the oviduct of a recipient female, or foster mother that has been induced to act as a recipient by mating with a vasectomized male. A major advantage of this method is its applicability to a wide variety of species

EMBRYONIC STEM CELL - MEDIATED GENE TRANSFER
This method involves: • • • • isolation of totipotent stem cells (stem cells that can develop into any type of specialized cell) from embryos the desired gene is inserted into these cells cells containing the desired DNA are incorporated into the host’s embryo, resulting in a chimeric animal Unlike the other 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. prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocyst stage of development.The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method. 6

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This technique is of particular importance for the study of the genetic control of developmental processes. This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination

RETROVIRUS MEDIATED GENE TRANSFER
Method involves: • • A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA . This method produces chimeras i.e. altered animals with mixed DNA.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 chimeras are inbred for as many as 20 generations until homozygous (carrying the desired transgene in every cell) transgenic offspring are born At this stage embryos carrying the transgene can be frozen and stored for subsequent implantation The method was successfully used in 1974 when a simian virus was inserted into mice embryos, resulting in mice carrying this DNA. To increase the probability of expression, gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid and Retroviruses are commonly used as vectors to transfer genetic material into the cell, taking advantage of their ability to infect host cells in this way. Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells. For any of these techniques the success rate in terms of live birth of animals containing the transgene is extremely low. Providing that the genetic manipulation does not lead to abortion, the result is a first generation (F1) of animals that need to be tested for the expression of the transgene. Depending on the technique used, the F1 generation may result in chimeras. At this stage embryos carrying the transgene can be frozen and stored for subsequent implantation

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At the end to conclude :.

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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.

PURPOSE OF TRANSGENIC ANIMALS :
• Contribution to human welfare
The benefits of these animals to human welfare can be grouped into areas:

1 . Agriculture 2 . Medicine 3 . Industry
The examples below are not intended to be complete but only to provide a sampling of the benefits. 1. Agricultural Applications • Transgenesis will allow larger herds with specific traits.

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. Scientists can improve the size of livestock genetically.

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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.

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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) Xeno-transplantation Patients die every year for lack of a replacement heart, liver, or kidney. 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 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

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nutritional or digestive needs.Rosie’s milk contains the human gene alphalactalbumin. c) 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.

3. 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.

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ETHICAL CONCERNS SURROUNDING TRANSGENESIS
This article focuses on the benefits of the technology; however, thoughtful ethical decision-making cannot be ignored by the biotechnology industry, scientists, policymakers, and the public. These ethical issues are : 1. Use of animals in biotechnological research causes great suffering to the animals. But most people seem to accept some animal suffering to serve the basic interest and welfare of mankind , this attitude has been termed as interest-sensitive speciesism. 2. It is felt that by using animals for the production of pharmaceutical proteins we reduce them to mere factories. This seems not to recognise that animals also are living beings which feel pleasure and pain just as we do.

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3. Some people feel that animals should be regarded as equal to humans in that they have the same basic rights as human beings. However, in most societies animals are relegated to a position several steps below that of man. 4. An argument attempts to focus on integrity of species in that each biological species has a right to exist as a separate identifiable entity. But biologists do not regard a species as a fixed, water-tight entity; rather they are regarded as dynamic, constantly evolving groups. 5. Finally, the introduction of human genes into animals, and vice versa, may be seen by many as clouding the definition of "humanness". But most of the known human genes are not unique, and comparable genes do occur in animals. In addition, many retroviruses have integrated into the human genome without any recognisable devaluation of our humanness. 6.It is unethical to modify an animal's genetic make-up for a specific purpose, without knowing if there will be any side-effects that will cause suffering to the animal. Conclusion: 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.

RELIGIOUS VIEWS OF TRANSGENIC ANIMALS :
Against transgenic animals: • • God laid down the structure of creation and any tampering with it is sinful. Manipulating DNA is manipulating 'life itself' - and this is tampering with something that God did not intend humanity to meddle with In favour of transgenic animals: • • As human beings have been given 'dominion' over the animals, they are entitled to tamper with them. Palaeontology shows that the structure of creation has changed over time as some species became extinct and new ones came into being. They say that this shows that there is nothing fixed about the structure of creation.

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TRANSGENIC ANIMAL AND RELIGIOUS FOOD LAWS:
• Transgenic animals pose problems for religions that restrict the foods that their species believers can eat, since they may produce animals that appear to be one species, but contain some elements of a forbidden.

SOME TRANSGENIC ANIMALS:

Gene transferred Cattle ,goat,sheep and swine – Human gene alpha 1 antitrypsin ,tissue plasminogen activator,blood clotting factor 9 ,and protein C Rabbits Human genes – interlukin2 ,growth harmone , tissue plasminogen activator , Bovine gene – alpha lactoglobulin Fish Salmon or rainbow trout growth harmone Anti-freeze protein gene ,alpha globin gene , chicken del crystalline protein gene etc. Mice

Objective

Gene farming

Acheivments Gene expressed in mammary tissues ; proteins secreted in milk in functional form

Remarks Experimental stages; alpha – trypsin recovered from tracey’s (a sheep)at the rate of 1.5kg per lactation. Experimental stages

gene farming

Gene expressed in mammary tissues ; proteins harvested from milk

Increased body growth

Upto 60% increase in size

Variable

Genes expressed in transgenic individuals

Transgenes stably inherited ,growth improved by selection .transgenes are stably integrated.

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human haemoglobin and specific circulating immunoglobulins(antibodies) Proteins from blood serum for blood transfusion and disease diagnosis

Genes expressed ;proteins released in blood serum

Experimental stages

SUMMARY
• Transgenic animals are those whose genetic material has been altered using genetic engineering techniques. These techniques are generally known as recombinant DNA technology.

• Process used
Firstly a suitable vector is chosen and the required gene is inserted into it i.e. recombinant DNA is produced. Then,this recombinant DNA will be introduced in the host cells (cultured cells) . selection of transformed ce lls and identification of the cell containing the gene (using reporter or marker gene).multiplication of the introduced gene in host. Techniques used: 1. DNA microinjection 2. retrovirus mediated gene transfer 3. embryonic stem cell – mediated gene transfer

their importance lies in all the fields – agricultural , industrial ,and in medicines

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considering them the creations of god and keeping in mind the issue of extinction of species there are many ethical issues surrounding transgenesis. There are number of transgenic animals produced in the past and many are in experimental stages as the research work is going on. A very dangerous consequence of transgenesis is the possibility of creating a destructive organism (chimera) ; kill of certain animals ; unintentional side effects.

REFERENCE

1.

Meyers , R.A. (ed. 1997) - Molecular biology and biotechnology : a comprehensive reference.

2.

Old , R.W. and Primrose , S.B.(ed.2001) – Principles of gene manipulations : an introduction to genetic engineering.

Internet links : http://www.actionbioscience.org/biotech • www.sciencedirect.com • Google > wikipedia >transgenic animals • www.users-rcn.com
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