Gene Cloning 2

8/2/2019 Gene Cloning 2

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Biology Project Work

Gene Cloning

Made By-Gaurav

Roll No.


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CertificateThis to certify that Master. Gauravof Class XIIth

Science has successfully completed the project on Gene

Cloning under the able guidance of her Biology teacher

M in the academic session 2011-2012.

Roll No.

Teachers Signature

Principles Signature

External Examiner


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Acknowledgement

This Particular Project has been one of the amiable and

aspiring assignments to me. I pay my thanks to our biology

teacher under whose guidance I was able to

complete the work. Lastly Im thankful to our principalMr.

Shekhar Jakhodiya who gave us the opportunity to conduct

this project.


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Content

Introduction

Different Types of Cloning

Steps of Gene CloningCloning techniques

Genetic Engineering

Reasons Why E.coli Is Used For Gene Cloning

Dolly -The Sheep

Species Cloned

Risks of Cloning

Should Human Be Cloned?

Applications of Gene Cloning

Conclusion

References


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Introduction

Genes

A gene is a short piece of DNA, which tells the body how to build a specific

protein, i.e., itcarry information that is necessary to make a protein. There are

approximately 30,000 genes in each cell of the human body. The combinationof all genes makes up the blueprint for the human body and its functions.

Cloning

Clonesare two or more organisms with identical genetic make-up derived, by

ASEXUAL REPRODUCTION, from a single common parent or ancestor, such as

identical twins.

Cloning refers to processes used to create copies ofDNAfragments (molecular

cloning), cells (cell cloning), ororganisms.

Hence,

Gene cloning is the act of making copies, orclones, of a single gene.Gene cloning provides opportunity to the scientists to study the structure and

function of genes in detail. For this purpose, gene of interest is inserted into the

bacterial cell which acts as a host. The cloned gene can be used for many

research purposes like detection of diseases, gene therapy and other medical

applications.
http://en.wikipedia.org/wiki/Cloninghttp://en.wikipedia.org/wiki/Cloninghttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Organismshttp://biotech.about.com/od/labtechniques/g/What-Is-A-Clone.htmhttp://biotech.about.com/od/labtechniques/g/What-Is-A-Clone.htmhttp://en.wikipedia.org/wiki/Organismshttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Cloning


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Different Types of Cloning

Cellular cloning

A useful tissue culture technique used to clone distinct lineages of cell lines

involves the use ofcloning rings (cylinders). According to this technique, a

single-cell suspension of cells that have been exposed to a mutagenic agentor

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 anearly 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 oftrypsin is added.

Cloned cells are collected from inside the ring and transferred to a new vessel

for further growth.

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. To amplify any DNA sequencein 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 allowprotein expression, tagging,

single strandedRNA and DNA production and a host of other manipulations.
http://en.wikipedia.org/w/index.php?title=Cloning_ring&action=edit&redlink=1http://en.wikipedia.org/wiki/Mutagenhttp://en.wikipedia.org/wiki/Selectionhttp://en.wikipedia.org/wiki/Polystyrenehttp://en.wikipedia.org/wiki/Trypsinhttp://en.wikipedia.org/wiki/Geneshttp://en.wikipedia.org/wiki/Promoter_(biology)http://en.wikipedia.org/wiki/Genetic_fingerprintinghttp://en.wikipedia.org/wiki/Genetic_fingerprintinghttp://en.wikipedia.org/wiki/Origin_of_replicationhttp://en.wikipedia.org/wiki/Cloning_vectorhttp://en.wikipedia.org/wiki/Protein_expressionhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/Protein_expressionhttp://en.wikipedia.org/wiki/Cloning_vectorhttp://en.wikipedia.org/wiki/Origin_of_replicationhttp://en.wikipedia.org/wiki/Genetic_fingerprintinghttp://en.wikipedia.org/wiki/Genetic_fingerprintinghttp://en.wikipedia.org/wiki/Promoter_(biology)http://en.wikipedia.org/wiki/Geneshttp://en.wikipedia.org/wiki/Trypsinhttp://en.wikipedia.org/wiki/Polystyrenehttp://en.wikipedia.org/wiki/Selectionhttp://en.wikipedia.org/wiki/Mutagenhttp://en.wikipedia.org/w/index.php?title=Cloning_ring&action=edit&redlink=1


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Organism cloning

Organism cloning (also called reproductive cloning) refers to the procedure ofcreating a new multicellular organism, genetically identical to another. In

essence this form of cloning is an asexual method of reproduction, where

fertilization or inter-gamete contact does not take place. Asexual reproduction

is a naturally occurring phenomenon in many species, including most plants

and some insects. Scientists have made some major achievements with cloning,

including the asexual reproduction of sheep and cows.

Human Cloning

Human cloning is the creation of a geneticallyidentical copy of an existing or

previously existing human. The term is generally used to refer

to artificial human cloning; human clones in the form ofidentical twins are

commonplace, with their cloning occurring during the natural process of

reproduction.

There are two commonly discussed types of human cloning: therapeutic

cloning and reproductivecloning.

Therapeutic cloning involves cloning adult cells for use in medicine and is an

active area of research.

Reproductive cloning would involve making cloned humans.

A third type of cloning calledreplacement cloning is a theoretical possibility,

and would be a combination of therapeutic and reproductive cloning.

Replacement cloning would entail the replacement of an extensively damaged,

failed, or failing body through cloning followed by whole or partialbrain

transplant.
http://en.wikipedia.org/wiki/Geneticshttp://en.wikipedia.org/wiki/Humanhttp://en.wikipedia.org/wiki/Identical_twinhttp://en.wikipedia.org/wiki/Brain_transplanthttp://en.wikipedia.org/wiki/Brain_transplanthttp://en.wikipedia.org/wiki/Brain_transplanthttp://en.wikipedia.org/wiki/Brain_transplanthttp://en.wikipedia.org/wiki/Identical_twinhttp://en.wikipedia.org/wiki/Humanhttp://en.wikipedia.org/wiki/Genetics


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Steps of Gene Cloning

Isolation of the Desired Gene: -

First step in gene cloning is the isolation of the desired part of DNA in which the

gene of interest is present. Similarly the bacterial plasmids are also isolated in

which the desired gene will be inserted. When the desired region with gene of

interest is identified, it is isolated by cutting it with restriction enzymes. As all

enzymes are proteins, restriction enzymes are also proteins which play a role of

catalysts in a chemical reaction. They cut the phosphodiester bond, a covalentbond of deoxyribose in DNA. There are specific sites on the DNA molecule called

restriction sites. These are the locations where gene of interest is isolated from

the rest of the DNA molecule by breaking the strand of DNA. Now this gene is

ready for the introduction to the vector.

Introduction of the Desired Gene into the Plasmid: -

When the gene of interest is isolated, it now needs a host cell where it can

replicate and can make multiple copies. For this purpose, plasmid is used which

is a molecule inside the bacterial cell. It has the ability to replicate out of the

bacterial chromosomal DNA. Plasmids are the best source of gene cloning as

they are able to replicate separately and independently from the bacteria's on

genetic material. They are double stranded circular molecules. When the gene

of the interest is inserted into the plasmid, the ring of the plasmid opens upgiving place to the gene to attach with it. Now the plasmid is ready for the

introduction into the host organisms as it contains a foreign gene.


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Insertion of Plasmid into the Host cell: -

Most commonly used plasmid is the Escherichia coli, specie of bacteria useful

for the human body. The plasmid with foreign gene is now inserted into the

host organism's body. Plasmid enters the host cell by two mechanisms; either it

is inserted by placing the cell incalcium chloride which will enable the plasmid

to enter the host cell or byElectroporation, in which through slight electric

current, pores will appear in the host cell's membrane, plasmid will enter

through these pores. Now the host cell is genetically modifies because it

contains the foreign within it.

Beginning of the Cloning of new Gene: -

As described above that plasmid is free of chromosomal DNA that is why when

it is inserted into the bacterial cell, it will start replicating at a very high speed

making identical copies of the desired gene. Scientists use E.coli as the host cell

for the plasmid, because it has the ability to replicate faster than any other

microorganism. It is the best microorganism used in recombinant DNA

technology.

Isolation of the Cloned gene: -

Scientists put the bacterial cell along with the culture into a culture, where it

replicates. Then the cloned gene is isolated again by using restriction enzymes

through the process known as lysis. Through this way, the cloned gene can be

isolated without any damage to the gene.


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Cloning Techniques

Embryo splitting involves dividing an eight cell embryo into single cells or

blastomeres. Transferring two of these blastomeres into an emptyzona

pellucida creates an embryo. Once the embryo reaches the blastocyst stage,

embryonic stem cells may be collected from the inner cell mass for use in

further research. A second option is to implant the embryo in a surrogate

mother, allowing it to develop fully. Offspring produced by embryo splitting will

be identical clones of one another if the embryo from which they were derived

is produced by fertilising an egg with sperm, the offspring will not be identicalto either of their genetic parents. This technique has been used successfully to

create non-human primate clones, but somatic cell nuclear transfer is the most

commonly used cloning technique.

In Intra-species cloning the nucleus of a somatic cell is taken from one

organism and placed in an enucleated egg from another member of the same

species. This method was used to produce the first cloned animal, Dolly the

sheep. It has subsequently been used to clone other species.

Inter-species cloning is more controversial than intra-species techniques

because the nucleus of a somatic cell from one organism is transferred to an

enucleated egg from a different species. Scientists have used this technique to

clone human cells by placing the nucleus of a human somatic cell in an

enucleated egg from a cow. The hybrid cells that were produced were able to

divide, but they were not permitted to develop to the blastocyst stage.
http://staff.um.edu.mt/acus1/FERTILIZATION_files/image002.jpghttp://staff.um.edu.mt/acus1/FERTILIZATION_files/image002.jpghttp://departments.weber.edu/chfam/Prenatal/images/Blastocyst.gifhttp://departments.weber.edu/chfam/Prenatal/images/Blastocyst.gifhttp://staff.um.edu.mt/acus1/FERTILIZATION_files/image002.jpghttp://staff.um.edu.mt/acus1/FERTILIZATION_files/image002.jpg


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Genetic engineering

It is the process of cloning genes into new organisms, or altering the DNAsequence to change the protein product. Genetic engineering depends on

our ability to perform the following essential procedures.

1. Polymerase Chain Reaction

The discovery of thermostable DNA polymerases, such as Taq Polymerase,

made it possible to manipulate DNA replication in the laboratory and was

essential to the development ofPCR. Primers specific to a particular region

of DNA, on either side of the gene of interest, are used, and replication is

stopped and started repetitively, generating millions of copies of that

gene. These copies can then be separated and purified using gel

electrophoresis.

2. Restriction Enzymes

The discovery ofenzymes known as restriction endonucleases has been

essential toprotein engineering . These enzymes cut DNA at specific

locations based on the nucleotide sequence. Hundreds of

differentrestriction enzymes, capable of cutting DNA at a distinct site,

have been isolated from many different strains of bacteria. DNA cut with a

restriction enzyme produces many smaller fragments, of varying sizes.

These can be separated using gel electrophoresis or chromatography.

3. Electrophoresis

Purifying DNA from a cell culture, or cutting it using restriction enzymes

wouldn't be of much use if we couldn't visualize the DNA - that is, find a

way to view whether or not your extract contains anything, or what size

fragments you've cut it in to. One way to do th is is by gel electrophoresis.

Gels are used for a variety of purposes, from viewing cut DNA to detecting

DNA inserts andknock outs.
http://biotech.about.com/od/glossary/g/What-Is-Dna.htmhttp://biotech.about.com/od/pcr/a/PCRtheory.htmhttp://biotech.about.com/od/glossary/g/PCRdef.htmhttp://biotech.about.com/od/proteinengineering/a/restrictenz.htmhttp://biotech.about.com/od/proteinengineering/a/restrictenz.htmhttp://biotech.about.com/od/glossary/g/Enzyme.htmhttp://biotech.about.com/od/technicaltheory/a/ProtEng.htmhttp://biotech.about.com/od/technicaltheory/a/ProtEng.htmhttp://biotech.about.com/od/technicaltheory/a/ProtEng.htmhttp://biotech.about.com/od/labtechniques/g/Electrophoresis.htmhttp://biotech.about.com/od/labtechniques/g/Electrophoresis.htmhttp://biotech.about.com/od/labtechniques/g/Electrophoresis.htmhttp://biotech.about.com/od/labtechniques/g/Electrophoresis.htmhttp://biotech.about.com/od/labtechniques/g/Knockout.htmhttp://biotech.about.com/od/labtechniques/g/Knockout.htmhttp://biotech.about.com/od/labtechniques/g/Knockout.htmhttp://biotech.about.com/od/labtechniques/g/Electrophoresis.htmhttp://biotech.about.com/od/labtechniques/g/Electrophoresis.htmhttp://biotech.about.com/od/technicaltheory/a/ProtEng.htmhttp://biotech.about.com/od/glossary/g/Enzyme.htmhttp://biotech.about.com/od/proteinengineering/a/restrictenz.htmhttp://biotech.about.com/od/glossary/g/PCRdef.htmhttp://biotech.about.com/od/pcr/a/PCRtheory.htmhttp://biotech.about.com/od/glossary/g/What-Is-Dna.htm


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4. Join Two Pieces of DNA

In genetic research it is often necessary to link two or more individual

strands of DNA, to create a recombinantstrand, or close a circular strand

that has been cut with restriction enzymes. Enzymes called DNA ligases can

create covalent bonds between nucleotide chains. The enzymes DNA

polymerase I and polynucl eot ide kinase are al so important in thi s process ,

for fi ll ing in gaps, or phosphorylat ing the 5' ends, respecti ve ly .

5. Selection of Small Self -Replicating DNA

Small circular pieces of DNA that are not part of a bacterial genome, but

are capable of self-replication, are known as plasmids. Plasmids are often

used as vectors to transport genes between microorganisms. In

biotechnology, once the gene of interest has been amplified and both the

gene and plasmid are cut by restriction enzymes, they are ligated together

generating what is known as a recombinant DNA. Viral (bacteriophage)

DNA can also be used as a vector, as can cosmids, recombinant plasmids

containing bacteriophage genes.

6. Method to Move a Vector into a Host Cell

The process of transferring genetic material on a vector such as a plasmid,

into new host cells, is called transformation. This technique requires that

the host cells are exposed to an environmental change which makes them

"competent" or temporarily permeable to the vector. Electroporation is

one such technique. The larger the plasmid, the lower the efficiency withwhich it is taken up by cells. Larger DNA segments are more easily cloned

using bacteriophage, retrovirus or other viral vectors or cosmids in a

method called transduction. Phage or viral vectors are often used

in regenerative medicine but may cause insertion of DNA in parts of our

chromosomes where we don't want it, causing complications and even

cancer.
http://biotech.about.com/od/glossary/g/Recombinant.htmhttp://biotech.about.com/od/proteintechnology/g/Vectors.htmhttp://biotech.about.com/od/labtechniques/g/Electroporation.htmhttp://biotech.about.com/od/faq/f/Retrovirus.htmhttp://biotech.about.com/od/glossary/g/regenerativemed.htmhttp://biotech.about.com/od/glossary/g/regenerativemed.htmhttp://biotech.about.com/od/faq/f/Retrovirus.htmhttp://biotech.about.com/od/labtechniques/g/Electroporation.htmhttp://biotech.about.com/od/proteintechnology/g/Vectors.htmhttp://biotech.about.com/od/glossary/g/Recombinant.htm


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7. Methods to Select Transgenic Organisms

Not all cells will take up DNA during transformation. It is essential that

there be a method of detecting the ones that do. Generally, plasmids carry

genes for antibiotic resistance and transgenic cells can be selected based

on expression of those genes and their ability to grow on media containing

that antibiotic. Alternative methods of selection depend on the prese nce of

otherreporter proteins such as the x-gal/lacZsystem, or green

fl uorescence protein, which al low selecti on based on color and

fl uorescence , respective ly.
http://biotech.about.com/od/labtechniques/g/transgenic.htmhttp://www.bio.davidson.edu/courses/genomics/method/reporters.htmlhttp://www.bio.davidson.edu/courses/genomics/method/reporters.htmlhttp://biotech.about.com/od/labtechniques/g/transgenic.htm


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Vectors

The vector serves as the carrier for the transfer or insertion of gene(s). Vectors

are among the essential tools for gene cloning.

eg - Agrobacterium tumefaciens, bacteriophages etc.
http://biotech.about.com/od/cloning/tp/DNAcloning.htmhttp://biotech.about.com/od/cloning/tp/DNAcloning.htm


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Top 5 Reasons E. coli is used for Gene Cloning

The microorganismEscherichia coli have a long history of use in thebiotechnology industry and is still the microorganism of choice for most gene

cloning experiments. AlthoughE. coli is known to the general population for

the infectious nature of one particular strain (0157:H7) few people are aware of

how versatile and usefulE. coliis to genetic research. There are several

reasonsE. colibecame so widely used and is still a common host for

recombinantDNA.

1. Genetic Simplicity

Bacteria make useful tools for genetic research because of their relativelysmall

genome sizecompared to eukaryotes.E. colicells only have about 4,400 genes

whereas the human genome projecthas determined that humans contain

approximately 30,000 genes. Also, bacteria, includingE. coli, live their entire

lifetime in a haploid state, with no second allele to mask the effects of

mutations during protein engineering experiments.

2. Growth Rate

Bacteria typicallygrow much fasterthan more complex organisms.E.

coligrows rapidly at a rate of one generation per twenty minutes under typical

growth conditions. This allows for preparation oflog-phase(mid-way to

maximum density) cultures overnight and genetic experimental results in mere

hours instead of several days, months or years. Faster growth also means

better production rates when cultures are used in scaled up fermentation

processes.
http://biotech.about.com/od/glossary/g/Recombinant.htmhttp://biotech.about.com/od/casestudies/a/humangenome.htmhttp://biotech.about.com/od/technicaltheory/a/ProtEng.htmhttp://biotech.about.com/od/glossary/g/Fermentation.htmhttp://biotech.about.com/od/glossary/g/Fermentation.htmhttp://biotech.about.com/od/glossary/g/Fermentation.htmhttp://biotech.about.com/od/technicaltheory/a/ProtEng.htmhttp://biotech.about.com/od/casestudies/a/humangenome.htmhttp://biotech.about.com/od/glossary/g/Recombinant.htm


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3. Safety

E. coliisnaturally foundin the intestinal tracts of humans and animals where it

helps provide nutrients (vitamins K and B12) to its host. There are many

different strains ofE. coli that may produce toxins or cause varying levels ofinfection if injested or allowed to invade other parts of the body. Despite the

bad reputation of one particularly toxic strain (O157:H7),E. coliare

generallyrelatively innocuous ifhandled with reasonable hygiene.

4. Conjugation and the Genome Sequence

TheE. coligenome was the first to be completely sequenced. Genetic mapping

inE. coliwas made possible by the discovery ofconjugation.E. coliis the most

highly studied microorganism and an advanced knowledge of its protein

expression mechanisms makes it simpler to use for experiments where

expression of foreign proteins and selection of recombinants is essential.

5. Ability to Host Foreign DNA

Most gene cloning techniques were developed using this bacterium and are still

more successful or effective inE. colithan in other microorganisms.E. coliis

readily transformed with plasmids and othervectors, easily

undergoes transduction, and preparation of competent cells (cells that will take

up foreign DNA) is not complicated. Transformations with other

microorganisms are often less successful.
http://biotech.about.com/od/glossary/g/conjugation.htmhttp://biotech.about.com/od/glossary/g/conjugation.htmhttp://biotech.about.com/od/proteintechnology/g/Vectors.htmhttp://biotech.about.com/od/proteintechnology/g/Vectors.htmhttp://biotech.about.com/b/2009/04/03/viral-vectors-for-transduction-not-transformation.htmhttp://biotech.about.com/b/2009/04/03/viral-vectors-for-transduction-not-transformation.htmhttp://biotech.about.com/b/2009/04/03/viral-vectors-for-transduction-not-transformation.htmhttp://biotech.about.com/od/proteintechnology/g/Vectors.htmhttp://biotech.about.com/od/glossary/g/conjugation.htm


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Dolly -The Sheep

Dolly (5 July 1996 14 February 2003) was a female domestic sheep, and the

firstmammalto be clonedfrom an adultsomatic cell, using the process of

nuclear transfer. She was cloned byIan Wilmut, Keith Campbelland colleagues

at the Roslin Institute nearEdinburgh in Scotland. She was born on 5 July 1996

and she lived until the age of six. The cell used as the donor for the cloning of

Dolly was taken from a mammary gland, and the production of a healthy clone

therefore proved that a cell taken from a specific part of the body could

recreate a whole individual.
http://en.wikipedia.org/wiki/Domestic_sheephttp://en.wikipedia.org/wiki/Mammalhttp://en.wikipedia.org/wiki/Cloninghttp://en.wikipedia.org/wiki/Somatic_cellhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Nuclear_transferhttp://en.wikipedia.org/wiki/Ian_Wilmuthttp://en.wikipedia.org/wiki/Keith_Campbell_(biologist)http://en.wikipedia.org/wiki/Roslin_Institutehttp://en.wikipedia.org/wiki/Edinburghhttp://en.wikipedia.org/wiki/Scotlandhttp://en.wikipedia.org/wiki/Mammary_glandhttp://en.wikipedia.org/wiki/File:Dolly_face_closeup.jpghttp://en.wikipedia.org/wiki/Mammary_glandhttp://en.wikipedia.org/wiki/Scotlandhttp://en.wikipedia.org/wiki/Edinburghhttp://en.wikipedia.org/wiki/Roslin_Institutehttp://en.wikipedia.org/wiki/Keith_Campbell_(biologist)http://en.wikipedia.org/wiki/Ian_Wilmuthttp://en.wikipedia.org/wiki/Nuclear_transferhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Somatic_cellhttp://en.wikipedia.org/wiki/Cloninghttp://en.wikipedia.org/wiki/Mammalhttp://en.wikipedia.org/wiki/Domestic_sheep


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Birth

Dolly was born 5 July 1996 to three mothers (one provided the egg; other DNA

and a third carried the cloned embryo to term). She was created using the

technique ofsomatic cell nuclear transfer, where the cell nucleusfrom an adultcell is transferred into an unfertilizedoocyte (developing egg cell) that has had

its nucleus removed. The hybrid cell is then stimulated to divide by an electric

shock, and when it develops into a blastocystit is implanted in a surrogate

mother. Dolly was the first clone produced from a cell taken from an adult

mammal. The production of Dolly showed that genes in the nucleus of such a

mature differentiatedsomatic cell are still capable of reverting back to an

embryonic totipotentstate, creating a cell that can then go on to develop into

any part of an animal. Dollys existence was announced to the public on 22

February 1997.

Life

Dolly lived for her entire life at the Roslin Institute in Edinburgh. There she was

bred with a Welsh Mountain ram and produced six lambs in total. Her first

lamb, named Bonnie, was born in April 1998. The next year Dolly produced twinlambs Sally and Rosie, and she gave birth to triplets Lucy, Darcy and Cotton in

the year after that. In the autumn of 2001, at the age of five, Dolly developed

arthritis and began to walk stiffly, but this was successfully treated with anti-

inflammatorydrugs.

Death

On 14 February 2003, Dolly was euthanisedbecause she had a progressive lung

disease and severe arthritis. A Finn Dorsetsuch as Dolly has a life expectancy

of around 11 to 12 years, but Dolly lived to be only six years of age. A post-

mortem examination showed she had a form oflung cancer called Jaagsiekte,

which is a fairly common disease of sheep and is caused by theretrovirus JSRV.
http://en.wikipedia.org/wiki/Somatic_cell_nuclear_transferhttp://en.wikipedia.org/wiki/Somatic_cell_nuclear_transferhttp://en.wikipedia.org/wiki/Somatic_cell_nuclear_transferhttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Oocytehttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Blastocysthttp://en.wikipedia.org/wiki/Cellular_differentiationhttp://en.wikipedia.org/wiki/Totipotencyhttp://en.wikipedia.org/wiki/Welsh_Mountain_sheephttp://en.wikipedia.org/wiki/Arthritishttp://en.wikipedia.org/wiki/Anti-inflammatoryhttp://en.wikipedia.org/wiki/Anti-inflammatoryhttp://en.wikipedia.org/wiki/Animal_euthanasiahttp://en.wikipedia.org/wiki/Finnish_Dorset_(sheep)http://en.wikipedia.org/wiki/Retrovirushttp://en.wikipedia.org/wiki/Retrovirushttp://en.wikipedia.org/wiki/JSRVhttp://en.wikipedia.org/wiki/JSRVhttp://en.wikipedia.org/wiki/JSRVhttp://en.wikipedia.org/wiki/Retrovirushttp://en.wikipedia.org/wiki/Finnish_Dorset_(sheep)http://en.wikipedia.org/wiki/Animal_euthanasiahttp://en.wikipedia.org/wiki/Anti-inflammatoryhttp://en.wikipedia.org/wiki/Anti-inflammatoryhttp://en.wikipedia.org/wiki/Arthritishttp://en.wikipedia.org/wiki/Welsh_Mountain_sheephttp://en.wikipedia.org/wiki/Totipotencyhttp://en.wikipedia.org/wiki/Cellular_differentiationhttp://en.wikipedia.org/wiki/Blastocysthttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Oocytehttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Somatic_cell_nuclear_transfer


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Cloning Of Dolly


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Species cloned

The modern cloning techniques involving nuclear transferhave been successfullyperformed on several species. Landmark experiments in chronological order:

Tadpole (1952): Many scientists questioned whether cloning had actually occurred

and unpublished experiments by other labs were not able to reproduce the reported

results.

Carp (1963): In China, embryologistTong Dizhouproduced the world's first cloned

fish by inserting the DNA from a cell of a male carp into an egg from a female carp.

He published the findings in a Chinese science journal.

Mice(1986: A mouse was the first mammal successfully cloned from an early

embryonic cell. Sovietscientists Chaylakhyan, Veprencev, Sviridova, and Nikitin had

the mouse "Masha" cloned. Research was published in the magazine "Biofizika"

volume II, issue 5 of 1987.

Sheep(1996): From early embryonic cells by Steen Willadsen. Megan and

Morag cloned from differentiated embryonic cells in June 1995 andDolly the

sheepfrom a somatic cell in 1997.

Rhesus Monkey: Tetra (January 2000) from embryo splitting

Gaur(2001): was the first endangered species cloned.

Cattle: Alpha and Beta (males, 2001) and (2005) Brazil

Cat: CopyCat"CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for

commercial reasons

Dog: Snuppy, a male Afghan houndwas the first cloned dog (2005).Rat: Ralph, the

first cloned rat (2003)

Mule: Idaho Gem, a john mule born 4 May 2003, was the first horse-family clone.

Horse: Prometea, a Haflinger female born 28 May 2003, was the first horse clone.

Water Buffalo: Samrupa was the first cloned water buffalo. It was born on February

6, 2009, atIndia's Karnal National Diary Research Institute but died five days later

due to lung infection.

Camel (2009): Injaz, is the first cloned camel.
http://en.wikipedia.org/wiki/Nuclear_transferhttp://en.wikipedia.org/wiki/Tadpolehttp://en.wikipedia.org/wiki/Carphttp://en.wikipedia.org/wiki/Chinahttp://en.wikipedia.org/wiki/Embryologisthttp://en.wikipedia.org/wiki/Tong_Dizhouhttp://en.wikipedia.org/wiki/Micehttp://en.wikipedia.org/wiki/Micehttp://en.wikipedia.org/wiki/Soviet_Unionhttp://en.wikipedia.org/wiki/Domestic_sheephttp://en.wikipedia.org/wiki/Domestic_sheephttp://en.wikipedia.org/wiki/Megan_and_Moraghttp://en.wikipedia.org/wiki/Megan_and_Moraghttp://en.wikipedia.org/wiki/Megan_and_Moraghttp://en.wikipedia.org/wiki/Dolly_the_sheephttp://en.wikipedia.org/wiki/Dolly_the_sheephttp://en.wikipedia.org/wiki/Rhesus_Monkeyhttp://en.wikipedia.org/wiki/Tetra_(monkey)http://en.wikipedia.org/wiki/Gaurhttp://en.wikipedia.org/wiki/Cattlehttp://en.wikipedia.org/w/index.php?title=Alpha_and_Beta&action=edit&redlink=1http://en.wikipedia.org/wiki/Cathttp://en.wikipedia.org/wiki/CC_(cat)http://en.wikipedia.org/wiki/Little_Nicky_(cat)http://en.wikipedia.org/wiki/Doghttp://en.wikipedia.org/wiki/Snuppyhttp://en.wikipedia.org/wiki/Afghan_houndhttp://en.wikipedia.org/wiki/Rathttp://en.wikipedia.org/wiki/Rathttp://en.wikipedia.org/w/index.php?title=Ralph_(cloned_rat)&action=edit&redlink=1http://en.wikipedia.org/wiki/Mulehttp://en.wikipedia.org/wiki/Idaho_Gemhttp://en.wikipedia.org/wiki/Horsehttp://en.wikipedia.org/wiki/Prometeahttp://en.wikipedia.org/wiki/Water_Buffalohttp://en.wikipedia.org/wiki/Samrupahttp://en.wikipedia.org/wiki/Indiahttp://en.wikipedia.org/wiki/Camelhttp://en.wikipedia.org/wiki/Injazhttp://en.wikipedia.org/wiki/Injazhttp://en.wikipedia.org/wiki/Camelhttp://en.wikipedia.org/wiki/Indiahttp://en.wikipedia.org/wiki/Samrupahttp://en.wikipedia.org/wiki/Water_Buffalohttp://en.wikipedia.org/wiki/Prometeahttp://en.wikipedia.org/wiki/Horsehttp://en.wikipedia.org/wiki/Idaho_Gemhttp://en.wikipedia.org/wiki/Mulehttp://en.wikipedia.org/w/index.php?title=Ralph_(cloned_rat)&action=edit&redlink=1http://en.wikipedia.org/wiki/Rathttp://en.wikipedia.org/wiki/Afghan_houndhttp://en.wikipedia.org/wiki/Snuppyhttp://en.wikipedia.org/wiki/Doghttp://en.wikipedia.org/wiki/Little_Nicky_(cat)http://en.wikipedia.org/wiki/CC_(cat)http://en.wikipedia.org/wiki/Cathttp://en.wikipedia.org/w/index.php?title=Alpha_and_Beta&action=edit&redlink=1http://en.wikipedia.org/wiki/Cattlehttp://en.wikipedia.org/wiki/Gaurhttp://en.wikipedia.org/wiki/Tetra_(monkey)http://en.wikipedia.org/wiki/Rhesus_Monkeyhttp://en.wikipedia.org/wiki/Dolly_the_sheephttp://en.wikipedia.org/wiki/Dolly_the_sheephttp://en.wikipedia.org/wiki/Megan_and_Moraghttp://en.wikipedia.org/wiki/Megan_and_Moraghttp://en.wikipedia.org/wiki/Domestic_sheephttp://en.wikipedia.org/wiki/Soviet_Unionhttp://en.wikipedia.org/wiki/Micehttp://en.wikipedia.org/wiki/Tong_Dizhouhttp://en.wikipedia.org/wiki/Embryologisthttp://en.wikipedia.org/wiki/Chinahttp://en.wikipedia.org/wiki/Carphttp://en.wikipedia.org/wiki/Tadpolehttp://en.wikipedia.org/wiki/Nuclear_transfer


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What are the risks of cloning?

Reproductive cloning is expensive and highly inefficient. More than 90% of cloning

attempts fail to produce viable offspring. More than 100 nuclear transfer procedures

could be required to produce one viable clone. In addition to low success rates, cloned

animals tend to have more compromised immune function and higher rates of

infection, tumor growth, and other disorders. Japanese studies have shown that

cloned mice live in poor health and die early. About a third of the cloned calves born

alive have died young, and many of them were abnormally large. Many cloned

animals have not lived long enough to generate good data about how clones age.

Appearing healthy at a young age unfortunately is not a good indicator of long-term

survival. Clones have been known to die mysteriously. For example, Australia's first

cloned sheep appeared healthy and energetic on the day she died, and the results

from her autopsy failed to determine a cause of death.

In 2002, researchers at the Whitehead Institute for Biomedical Research in

Cambridge, Massachusetts, reported that the genomes of cloned mice are

compromised. In analyzing more than 10,000 liver and placenta cells of cloned mice,they discovered that about 4% of genes function abnormally. The abnormalities do

not arise from mutations in the genes but from changes in the normal activation or

expression of certain genes.

Problems also may result from programming errors in the genetic material from a

donor cell. When an embryo is created from the union of a sperm and an egg, the

embryo receives copies of most genes from both parents. A process called

"imprinting" chemically marks the DNA from the mother and father so that only one

copy of a gene (either the maternal or paternal gene) is turned on. Defects in the

genetic imprint of DNA from a single donor cell may lead to some of the

developmental abnormalities of cloned embryos.


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Should humans be cloned?

Due to the inefficiency of animal cloning (only about 1 or 2 viable offspring for

every 100 experiments) and the lack of understanding about reproductive

cloning, many scientists and physicians strongly believe that it would be

unethical to attempt to clone humans. Not only do most attempts to clone

mammals fail, about 30% of clones born alive are affected with "large-offspringsyndrome" and other debilitating conditions. Several cloned animals have died

prematurely from infections and other complications. The same problems

would be expected in human cloning. In addition, scientists do not know how

cloning could impact mental development. While factors such as intellect and

mood may not be as important for a cow or a mouse, they are crucial for the

development of healthy humans. With so many unknowns concerning

reproductive cloning, the attempt to clone humans at this time is considered

potentially dangerous and ethically irresponsible.


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Applications of Gene Cloning

The process of gene cloning can be used for producing eukaryotic proteins inthe bacterial cells, and for establishing a gene library. Following are some of

the fields where gene cloning has found its application.

Medical Utility

In medicine, bacteria play a vital role for the synthesis or production of many

vitamins, hormones and antibiotics. It is done by introducing the desired geneinto the plasmid and then this plasmid replicates to make multiple copies of this

gene. If scientists have to treat some dangerous disease, they clone the healthy

gene and then insert it into the organism to replace it with the diseased gene.

The ability to clone a gene is not only valuable for conducting biological

research. Many important pharmaceutical drugs and industrial enzymes are

produced from cloned genes. For example, insulin, clotting factors, human

growth hormone, cytokines (cell growth stimulants), and several anticancerdrugs in use are produced from cloned genes

Agricultural Utility

Bacteria also are important for the nitrogen fixation. Bacteria along with the

desired gene are used to increase the crop productivity and health. They make

the farmers free of using expensive fertilizers which can damage the crops.


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Conclusion

Gene cloning is the replication of DNA fragments by the use of a self-replicating

genetic material. Gene cloning duplicates only individual genes of an

organism's DNA.

Deoxyribonucleic acid, or DNA, is the genetic material contained within all

organisms.

Specific genes are isolated on the DNA molecule for cloning through the use of

restrictive enzymes.

Plasmids are the self-replicating genetic material that actually duplicates the

host DNA fragment. Plasmids are biologically engineered to be compatible with

the host DNA fragment.

Cloningdoes not introduce new genes into a population so will not increase

genetic diversity. Cloning of domesticated animals could be important in the

future production oftransgenic livestock.

Cloning may have uses inpreserving endangered species and may become a

viable tool forreviving extinct species.
http://en.wikipedia.org/wiki/Genetic_diversityhttp://en.wikipedia.org/wiki/Genetically_modified_organismhttp://en.wikipedia.org/wiki/Extinctionhttp://en.wikipedia.org/wiki/Extinctionhttp://en.wikipedia.org/wiki/Extinctionhttp://en.wikipedia.org/wiki/Genetically_modified_organismhttp://en.wikipedia.org/wiki/Genetic_diversity


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References

www.google.com

www.ornl.gov.in

www.wikipedia.org

www.ehow.com

www.medicine.jrank.org

www.lukashensel.de.com

www.biotech.about.com

www.ncbi.nlm.nih.gov

www.tutorvista.com

www.departments.weber.edu
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Documents

Gene Cloning 2