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GENETIC

ENGINEERING

HARIPREM TAMILCHELVAN

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INTRODUCTION……….1

HISTORY………..……….2

PROCESS OF GENETIC

ENGINEERING………….4

APPLICATION…………….9

CONCLUSION……….…16

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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

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INTRODUCTION

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he origins of biotechnology culminated with the birth of genetic engineering.

Genetic engineering based on genetics, a science started form the early 1900’s 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 1970’s, 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, Genentech’s 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.

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HISTORY

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The People’s 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.

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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.

PROCESS OF GENETIC ENGINEERING

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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

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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 T-

DNA. 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.

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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.

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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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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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APPLICATION

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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

Example Genetic Change

Herbicide tolerance Soybean 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

Insect resistance Corn Resistance to insect pests, specifically the European corn

borer, through expression of the insecticidal protein

Cry1Ab from Bacillus thuringiensis

Altered fatty acid

composition

Canola High laurate levels achieved by inserting the gene for

ACP thioesterase from the California bay tree

Umbellularia californica

Virus resistance Plum Resistance to plum pox virus conferred by insertion of a

coat protein (CP) gene from the virus

Vitamin enrichment 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

Vaccines Tabacco Hepatitis B virus surface antigen (HBsAg) produced in

transgenic tobacco induces immune response when

injected into mice

Oral vaccines Maize Fusion protein (F) from Newcastle disease virus (NDV)

expressed in corn seeds induces an immune response

when fed to chickens

Faster maturation 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% that’s 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 forensics—from

verifying paternity to identifying murder suspects—this 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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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.

CONCLUSION

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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-could-

rovidetreatment- 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)

REFERENCE

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14. http://www.nature.com/scitable/topicpage/genetically-modified-organisms-gmos-

transgenic-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