GENETIC ENGINEERING

HARIPREM TAMILCHELVAN

111091 06227 - 010

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- C O N T- E - N - T
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INTRODUCTION.1 HISTORY...2 PROCESS OF GENETIC ENGINEERING.4 APPLICATION.9 CONCLUSION.16

INTRODUCTION

enetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest.

The introduction of new DNA does not require the use of classical genetic methods; however traditional breeding methods are typically used for the propagation of recombinant organisms. The most common form of genetic engineering involves the insertion of new genetic material at an unspecified location in the host genome. This is accomplished by isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence containing the required genetic elements for expression, and then inserting this construct into the host organism. Other forms of genetic engineering include gene targeting and knocking out specific genes via engineered nucleases such as zinc finger nucleases or engineered homing endonucleases. Genetic engineering techniques have been applied in numerous fields including research, biotechnology, and medicine. Medicines such as insulin and human growth hormone are now produced in bacteria, experimental mice such as the oncomouse and the knockout mouse are being used for research purposes and insect resistant and/or herbicide tolerant crops have been commercialized. Genetically engineered plants and animals capable of producing biotechnology drugs more cheaply than current methods

HISTORY

he origins of biotechnology culminated with the birth of genetic engineering. Genetic engineering based on genetics, a science started form the early 1900s based on experiments by the Austrian monk, Gregor Mendel.

In 1944, DNA is identified as the carrier of genetic information by Oswald Avery Colin

McLeod and Maclyn McCarty. Later two important key events happened. One was the 1953 discovery of the structure of DNA, by Watson and Crick, and the other was the 1973 discovery by Cohen and Boyer of a recombinant DNA technique by which a section of DNA was cut from the plasmid of an E. coli bacterium and transferred into the DNA of another. During the late 1970s, researchers used recombinant DNA to engineer bacteria to produce small quantities of insulin and interferon. One of the key scientific figures that attempted to highlight the promising aspects of genetic engineering was Joshua Lederberg, a Stanford professor and Nobel laureate. In 1980, green genetic engineering was born. Genetic material is introduced into cell cultures for the first time ever with the aid of Agrobacterium tumefaciens. In 1982, The U.S Food and Drug Administration approve the first genetically engineered drug, Genentechs Humulin, a form of human insulin produced by bacteria. In 1987, the first field tests of genetically engineered crops (tobacco and tomato) are conducted in the United States. Committee of the national Academy of Sciences concluded that transferring genes between species of organisms posed no serious environmental hazards.

The Peoples Republic of China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992. In 1994 Calgene attained approval to commercially release the Flavr Savr tomato, a tomato engineered to have a longer shelf life. In 1994, the European Union approved tobacco engineered to be resistant to the herbicide bromoxynil, making it the first genetically engineered crop commercialized in Europe.] In 1995, Bt Potato was approved safe by the Environmental Protection Agency, making it the first pesticide producing crop to be approved in the USA. In 2009 11 transgenic crops were grown commercially in 25 countries, the largest of which by area grown were the USA, Brazil, Argentina, India, Canada, China, Paraguay and South Africa. In 2010, scientists at the J. Craig Venter Institute announced that they had created the first synthetic bacterial genome, and added it to a cell containing no DNA. The resulting bacterium, named Synthia, was the world's first synthetic life form.

PROCESS OF GENETIC ENGINEERING

There are 7 steps in genetic engineering 1. Isolating the gene 2. Constructs 3. Gene targeting 4. Transformation 5. Selection 6. Regeneration 7. Confirmation

ISOLATING THE GENE The gene to be inserted into the genetically modified organism must be chosen and isolated. Presently, most genes transferred into plants provide protection against insects or tolerance to herbicides. In animals the majority of genes used are growth hormone genes. Once chosen the genes must be isolated. This typically involves multiplying the gene using polymerase chain reaction (PCR). If the chosen gene or the donor organism's genome has been well studied it may be present in a genetic library. If the DNA sequence is known, but no copies of the gene are available, it can be artificially synthesized. Once isolated, the gene is inserted into a bacterial plasmid.

CONSTRUCT The gene to be inserted into the genetically modified organism must be combined with other genetic elements in order for it to work properly. The gene can also be modified at this stage for better expression or effectiveness. As well as the gene to be inserted most constructs contain a promoter and terminator region as well as a selectable marker gene. The promoter region initiates transcription of the gene and can be used to control the location and level of gene expression, while the terminator region ends transcription. The selectable marker, which in most cases confers antibiotic resistance to the organism it is expressed in, is needed to determine which cells are transformed with the new gene. The constructs are made using recombinant DNA techniques, such as restriction digests, ligations and molecular cloning.

GENE TARGATING The most common form of genetic engineering involves inserting new genetic material randomly within the host genome. Other techniques allow new genetic material to be inserted at a specific location in the host genome or generate mutations at desired genomic loci capable of knocking out endogenous genes. The technique of gene targeting uses homologous recombination to target desired changes to a specific endogenous gene. This tends to occur at a relatively low frequency in plants and animals and generally requires the use of selectable markers. The frequency of gene targeting can be greatly enhanced with the use of engineered nucleases such as zinc finger nucleases, engineered homing endonucleases, or nucleases created from TAL effectors. In addition to enhancing gene targeting, engineered nucleases can also be used to introduce mutations at endogenous genes that generate a gene knockout

TRANSFORMATION About 1% of bacteria are naturally able to take up foreign DNA but it can also be induced in other bacteria. Stressing the bacteria for example, with a heat shock or an electric shock, can make the cell membrane permeable to DNA that may then incorporate into their genome or exist as extrachromosomal DNA. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cells nuclear envelope directly into the nucleus or through the use of viral vectors. In plants the DNA is generally inserted using Agrobacterium-mediated recombination or biolistics. In Agrobacterium-mediated recombination the plasmid construct must also contain TDNA. Agrobacterium naturally inserts DNA from a tumor inducing plasmid into any susceptible plant's genome it infects, causing crown gall disease. The T-DNA region of this plasmid is responsible for insertion of the DNA. The genes to be inserted are cloned into a binary vector, which contains T-DNA and can be grown in both E. Coli and Agrobacterium. Once the binary vector is constructed the plasmid is transformed into Agrobacterium containing no plasmids and plant cells are infected. The Agrobacterium will then naturally insert the genetic material into the plant cells. In biolistics particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will enter the cells and transform them. This method can be used on plants that are not susceptible to Agrobacterium infection and also allows transformation of plant plastids. Another transformation method for plant and animal cells is electroporation. Electroporation involves subjecting the plant or animal cell to an electric shock, which can make the cell membrane permeable to plasmid DNA. In some cases the electroporated cells will incorporate the DNA into their genome.

SELECTION Not all the organism's cells will be transformed with the new genetic material; in most cases a selectable marker is used to differentiate transformed from untransformed cells. If a cell has been successfully transformed with the DNA it will also contain the marker gene. By growing the cells in the presence of an antibiotic or chemical that selects or marks the cells expressing that gene it is possible to separate the transgenic events from the non-transgenic. Another method of screening involves using a DNA probe that will only stick to the inserted gene. A number of strategies have been developed that can remove the selectable marker from the mature transgenic plant.

REGENERATION As often only a single cell is transformed with genetic material the organism must be regrown from that single cell. As bacteria consist of a single cell and reproduce clonally regeneration is not necessary. In plants this is accomplished through the use of tissue culture. Each plant species has different requirements for successful regeneration through tissue culture. If successful an adult plant is produced that contains the transgene in every cell. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. When the offspring is produced they can be screened for the presence of the gene. All offspring from the first generation will be heterozygous for the inserted gene and must be mated together to produce a homozygous animal.

CONFORMATION The finding that a recombinant organism contains the inserted genes is not usually sufficient to ensure that the genes will be expressed in an appropriate manner in the intended tissues of the recombinant organism. To examine the presence of the gene, further analysis frequently uses PCR, Southern hybridization, and DNA sequencing, which serve to determine the chromosomal location and copy number of the inserted gene. To examine expression of the trans-gene, an extensive analysis of transcription, RNA processing patterns, and the expression and localization of the protein product(s) is usually necessary, using methods including northern hybridization, quantitative RT-PCR, Western blot, immunofluorescence and phenotypic analysis. When appropriate, the organism's offspring are studied to confirm that the trans-gene and associated phenotype are stably inherited.

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APPLICATION
GENETICALLY MODIFIED FOOD AND CROPS

ne major application of genetic engineering techniques is in the realm of food production. With the world population expanding and synthetic pesticides decreasing in effectiveness, novel solutions are increasingly in demand.

Genetically modified foods are one such solution. Genetically modified foods can: increase plants' resistance to pesticides and herbicides, thereby decreasing the need for these pollutant chemicals; allow plants to manufacture their own pesticides to ward off insects; increase the yields of many staple crops and thereby ward off starvation in many areas of the world; and allow plants to grow under adverse weather conditions or in poor soil, thereby increasing the amount of arable land on the planet. Genetic modification involves the insertion or deletion of genes. In the process of cisgenesis, genes are artificially transferred between organisms that could be conventionally bred. In the process of transgenesis, genes from a different species are inserted, which is a form of horizontal gene transfer. In nature this can occur when exogenous DNA penetrates the cell membrane for any reason. To do this artificially may require transferring genes as part of an attenuated virus genome or physically inserting the extra DNA into the nucleus of the intended host using a microsyringe, or as a coating on gold nanoparticles fired from a gene gun. The method to introduce new genes into plants requires several important factors such as specific promoter, codon usage of the gene and how to deactivate the gene. The specific
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promoter must pertain to area that we want the gene to express. For instance, if we want the gene to express only in the rice instead of the leaf than we would only use an endosperm specific promoter. The reason is because we only want our transgenic genes to express only in the rice and not the leaves. The codon usage of the gene must also be more optimize for the rice since there are several different codons for each of the 20 amino acid. The transgenic genes should also be able to be denatured by heat in order for human consumption. Examples of GMOs Resulting from Agricultural Biotechnology Genetically Conferred Trait Herbicide tolerance Example Soybean Genetic Change

Insect resistance

Altered fatty acid composition Virus resistance Vitamin enrichment

Vaccines

Oral vaccines

Faster maturation

Glyphosate herbicide (Roundup) tolerance conferred by expression of a glyphosate-tolerant form of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) isolated from the soil bacterium Agrobacterium tumefaciens, strain CP4 Corn Resistance to insect pests, specifically the European corn borer, through expression of the insecticidal protein Cry1Ab from Bacillus thuringiensis Canola High laurate levels achieved by inserting the gene for ACP thioesterase from the California bay tree Umbellularia californica Plum Resistance to plum pox virus conferred by insertion of a coat protein (CP) gene from the virus Rice Three genes for the manufacture of beta-carotene, a precursor to vitamin A, in the endosperm of the rice prevent its removal (from husks) during milling Tabacco Hepatitis B virus surface antigen (HBsAg) produced in transgenic tobacco induces immune response when injected into mice Maize Fusion protein (F) from Newcastle disease virus (NDV) expressed in corn seeds induces an immune response when fed to chickens Coho salmon A type 1 growth hormone gene injected into fertilized fish eggs results in 6.2% retention of the vector at one year of age, as well as significantly increased growth rates

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CLONNING

loning is the production of multiple, identical offspring. A clone is an animal who is genetically identical to its donor "parent". We now know that this can be achieved using cells derived from a microscopic embryo, a fetus, or from an adult animal.

Cloning from adult animals was introduced to the public in 1997 when scientists announced the birth of Dolly, the first animal cloned in this way. There have now been hundreds of clones produced from skin cells taken from adult sheep, cattle, goats, pigs and mice. The real key to cloning an adult animal is the ability to reprogram the skin cell nucleus and cause it to begin developing as if it was a newly fertilized egg. Cloning requires specialized microsurgery tools and involves five basic steps: Enucleation of the recipient egg Transfer of the donor cell into the recipient egg Fusion of the donor cell to the recipient egg Culturing the resulting cloned embryo in the incubator Transferring the developing embryo into the reproductive tract of a surrogate mother Dolly the sheep may have been the world's most famous clone, but she was not the first. Cloning creates a genetically identical copy of an animal or plant. Many animals - including frogs, mice, and cows - had been cloned before Dolly. Plants are often cloned taking a cutting produces a clone of the original plant. Human identical twins are also clones. Dolly was the first mammal to be cloned from an adult cell, rather than an embryo. This was a major scientific achievement, but also raised ethical concerns.

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Since 1996, when Dolly was born, other sheep have been cloned from adult cells, as have mice, rabbits, horses and donkeys, pigs, goats and cattle. In 2004 a mouse was cloned using a nucleus from an olfactory neuron, showing that the donor nucleus can come from a tissue of the body that does not normally divide. Process of Cloning

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DNA FINGERPRINTING

NA profiling (also called DNA testing, DNA typing, or genetic fingerprinting) is a technique employed by forensic scientists to assist in the identification of individuals by their respective DNA profiles. DNA profiles are encrypted sets of

numbers that reflect a person's DNA makeup, which can also be used as the person's identifier. DNA profiling should not be confused with full genome sequencing. It is used in, for example, parental testing and criminal investigation. DNA sequences are extremely long, and comparing an entire DNA sequence with another would be hard to do. Fortunately, though, about 99% of human DNA is identical from one person to the next. The 1% thats different includes several frequently repeating sequences; the number of repeating sequences in any given position on a chromosome is different for each person. Therefore, in DNA fingerprinting, fragments of DNA are extracted and a collection is created that is unique for each person. There are several techniques for doing so; they differ mainly in how the fragments are extracted and how they are converted into a form that can be analyzed for identification. While human DNA fingerprinting has numerous uses in law and forensicsfrom verifying paternity to identifying murder suspectsthis technique also applies to other organisms. Plants, animals, and even bacteria have unique DNA fingerprints. An increasing range of applications makes use of this fact.

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Process of DNA Fingerprinting

Examples :

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MEDICINE

ome of the most promising and powerful applications of genetic engineering are in the field of medicine. Researchers are using it to diagnose and predict disease, and to develop therapies and drugs to treat devastating diseases like cancer, Alzheimer's, diabetes, and cystic fibrosis. Explore more about ways genetic engineering techniques can be used for medical purposes.

Recombinant DNA Recombinant DNA is one of the core techniques of genetic engineering. It is the process of removing DNA from one organism and inserting it into the DNA of another organism, giving it new traits. Recombinant DNA can be used to make crops resistant to pests or disease, it can be used to make livestock leaner or larger. In medicine, the technique can be used to develop drugs, vaccines, and to reproduce important human hormones and proteins. By engineering human DNA into a host organism, that organism can be turned into a factory for important medical products. Insulin production is an excellent example of the recombinant DNA process. Host organisms can range from bacteria like E. coli, to plants, to animals. Genetically Engineered Pharmaceuticals insulin for diabetics factor VIII for males suffering from hemophilia A factor IX for hemophilia B human growth hormone (GH) erythropoietin (EPO) for treating anemia three types of interferons - fight viral infections several interleukins granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating the bone marrow after a bone marrow transplant tissue plasminogen activator (TPA) for dissolving blood clots adenosine deaminase (ADA) for treating some forms of severe combined immunodeficiency (SCID) angiostatin and endostatin for trials as anti-cancer drugs parathyroid hormone

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CONCLUSION
Genetic engineering has the potential to transform our lives in many positive ways. Rejection of this new technology on the ground that it is unnatural or inherently immoral is unwarranted and seems to be based on little more than an instinctive adverse reaction. Biotechnology is an extension of already accepted and well-established techniques, such as directed breeding, but with the distinct advantage of producing more predictable and more rapid results. There are risks involved with this new technology, but provided that it is appropriately regulated, its potential benefits outweigh its harms. Legislators and other responsible decision-makers should not implement regulations that unduly restrict implementation of genetic engineering. In particular, existing mechanisms that ensure the safety of testing protocols should be sufficient for somatic genetic therapies for humans. With respect to germline enhancements for plants and animals, we recommend a better coordinated effort among relevant regulatory agencies, such as the Food and Drug Administration and the Department of Agriculture, to ensure there are no gaps in the regulatory framework. Enhanced organisms should be rigorously evaluated and tested in isolated conditions prior to their release in the wild. Germline alterations for humans should not be prohibited outright, certainly not in advance of their availability. However, given the special risks posed by human germline alterations, each proposed alteration needs to be carefully evaluated, not just with respect to immediate benefits and harms, but also with respect to the effects that the proposed alteration may have on our social structure and the distribution of social goods.

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REFERENCE
1. http://en.wikipedia.org/wiki/Genetic_engineering 2. Ruiz-Marrero, Carmelo. 2002. Genetic Pollution: Biotech Corn Invades Mexico. Available at http://www.corpwatch.org/article.php?id=2088. 3. Rebelo, Paulo. 2004. GM Cow Milk Could Provide Treatment for Blood Disease. Available at http://www.scidev.net/content/news/eng/gm-cow-milk-couldrovidetreatment- for-blood-disease.cfm. 4. Epstein, Ron. 1999. Ethical Dangers of Genetic Engineering. Institute for World Religions. Available at http://www.greens.org/s-r/20/20-01.html 5. http://www2.ellendale.k12.nd.us/hsmain/thoffman/biology/geneticengineeringwebquestpr esentation.pdf 6. file:///C:/Users/syanmugam/Desktop/Genetic%20Engineering/Applications%20of%20Ge netic%20Engineering.htm 7. http://geneticengineeringmedicine.com 8. http://www.iptv.org/exploremore/ge/uses/use2_medical.cfm 9. http://www.eplantscience.com/botanical_biotechnology_biology_chemistry/biotechnolog y/genes_genetic_engineering/genetic_engineering_for_human_welfare/biotech_methods _of_dna_profiling.php 10. http://en.wikipedia.org/wiki/DNA_profiling 11. http://itotd.com/articles/572/dna-fingerprinting 12. http://www.animalresearch.info/en/medical/timeline/Dolly 13. http://en.wikipedia.org/wiki/Dolly_(sheep)

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14. http://www.nature.com/scitable/topicpage/genetically-modified-organisms-gmostransgenic-crops-and-732 15. http://en.wikipedia.org/wiki/Genetically_modified_food 16. http://www.foe.co.uk/resource/briefings/gm_crops_food.pdf 17. http://www.bionetonline.org/english/content/ff_cont3.htm 18. Pre-U Text STPM Longman Biology Volume 1 and 2

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