Biology Project Work

Gene Cloning

Made By-Gaurav Roll No.

Certificate

This to certify that Master. Gaurav of 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.

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Teachers Signature

Principles Signature

External Examiner

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 principal Mr. Shekhar Jakhodiya who gave us the opportunity to conduct this project.

Content

Introduction Different Types of Cloning Steps of Gene Cloning Cloning 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

Introduction
Genes
A gene is a short piece of DNA, which tells the body how to build a specific protein, i.e., it carry information that is necessary to make a protein. There are approximately 30,000 genes in each cell of the human body. The combination of all genes makes up the blueprint for the human body and its functions.

Cloning
Clones are 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 of DNA fragments (molecular cloning), cells (cell cloning), or organisms.

Hence, Gene cloning is the act of making copies, or clones, 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.

Different Types of Cloning

Cellular cloning
A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders). According to this technique, a single-cell suspension of cells that have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies; each arising from a single and potentially clonal distinct cell. At an early growth stage when colonies consist of only a few of cells, sterile polystyrene rings (cloning rings), which have been dipped in grease are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.

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 sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations.

Organism cloning
Organism cloning (also called reproductive cloning) refers to the procedure of creating 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 genetically identical copy of an existing or previously existing human. The term is generally used to refer to artificial human cloning; human clones in the form of identical 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 reproductive cloning. 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 called replacement 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 partial brain transplant.

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 covalent bond 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 up giving 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.

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 in calcium chloride which will enable the plasmid to enter the host cell or by Electroporation, 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.

Cloning Techniques
Embryo splitting involves dividing an eight cell embryo into single cells or blastomeres. Transferring two of these blastomeres into an empty zona 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 identical to 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.

Genetic engineering
It is the process of cloning genes into new organisms, or altering the DNA sequence 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 Poly merase, made it possible to manipulate DNA replication in the laboratory and was essential to the development of PCR. 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 of enzymes known as restriction endonucleases has been essential to protein engineering. These enzymes cut DNA at specific locations based on the nucleotide sequence. Hundreds of different restriction 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 into. One way to do this is by gel electrophoresis. Gels are used for a variety of purposes, from viewing cut DNA to detecting DNA inserts and knock outs.

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 recombinant strand, 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 polynucleotide kinase are also important in this process, for filling in gaps, or phosphorylating the 5' ends, respectively.

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 with which 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.

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 other reporter proteins such as the x-gal/ lacZ system, or green fluorescence protein, which allow selection based on color and fluorescence, respectively.

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.

Top 5 Reasons E. coli is used for Gene Cloning

The microorganism Escherichia coli have a long history of use in the biotechnology industry and is still the microorganism of choice for most gene cloning experiments. Although E. 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 useful E. coli is to genetic research. There are several reasons E. coli became so widely used and is still a common host for recombinant DNA.

1. Genetic Simplicity Bacteria make useful tools for genetic research because of their relatively small genome size compared to eukaryotes. E. coli cells only have about 4,400 genes whereas the human genome project has determined that humans contain approximately 30,000 genes. Also, bacteria, including E. 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 typically grow much faster than more complex organisms. E. coli grows rapidly at a rate of one generation per twenty minutes under typical growth conditions. This allows for preparation of log-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.

3. Safety E. coli is naturally found in the intestinal tracts of humans and animals where it helps provide nutrients (vitamins K and B12) to its host. There are many different strains of E. coli that may produce toxins or cause varying levels of infection if injested or allowed to invade other parts of the body. Despite the bad reputation of one particularly toxic strain (O157:H7), E. coli are generally relatively innocuous if handled with reasonable hygiene. 4. Conjugation and the Genome Sequence The E. coli genome was the first to be completely sequenced. Genetic mapping in E. coli was made possible by the discovery of conjugation. E. coli is 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 in E. coli than in other microorganisms. E. coli is readily transformed with plasmids and other vectors, 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.

Dolly -The Sheep

Dolly (5 July 1996 14 February 2003) was a female domestic sheep, and the first mammal to be cloned from an adult somatic cell, using the process of nuclear transfer. She was cloned by Ian Wilmut, Keith Campbell and colleagues at the Roslin Institute near Edinburgh 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.

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 of somatic cell nuclear transfer, where the cell nucleus from an adult cell is transferred into an unfertilized oocyte (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 blastocyst it 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 differentiated somatic cell are still capable of reverting back to an embryonic totipotent state, 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 twin lambs 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 antiinflammatory drugs.

Death On 14 February 2003, Dolly was euthanised because she had a progressive lung disease and severe arthritis. A Finn Dorset such as Dolly has a life expectancy of around 11 to 12 years, but Dolly lived to be only six years of age. A postmortem examination showed she had a form of lung cancer called Jaagsiekte, which is a fairly common disease of sheep and is caused by the retrovirus JSRV.

Cloning Of Dolly

Species cloned
The modern cloning techniques involving nuclear transfer have been successfully performed 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, embryologist Tong Dizhou produced 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. Soviet scientists 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 and Dolly the sheep from 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 hound was 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, at India's Karnal National Diary Research Institute but died five days later due to lung infection. Camel (2009): Injaz, is the first cloned camel.

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.

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-offspring syndrome" 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.

Applications of Gene Cloning

The process of gene cloning can be used for producing eukaryotic proteins in the 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 gene into 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 anticancer drugs 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.

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. Cloning does not introduce new genes into a population so will not increase genetic diversity. Cloning of domesticated animals could be important in the future production of transgenic livestock. Cloning may have uses in preserving endangered species and may become a viable tool for reviving extinct species.

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