The topic of cloning came to the fore in March 1997 when Dr.

Ian Wilmut announced that "Dolly" the sheep had been born at the Roslin Institute in Scotland. Dolly was a clone, genetically identical to her cell-donor. (This is similar to the movie "Jurassic Park," where a drop of preserved dinosaur blood was used to generate new generations of dinosaurs.) Oregon researchers have since announced their cloning of monkeys, and scientists predict that human cloning is achievable within the next decade. It is important to establish up-front how cloning works, and what exactly it is and isn't. Otherwise, the topic can quickly turn into a far-out discussion of science fiction, which may be boring for anyone who's not a Trekkie. Here's the key background information. Every cell in our body contains the genetic coding of our entire physical make-up. For example, a liver cell contains all the genetic coding for eye color, hair color, intelligence, skin tone, etc. What happens during the process of fetal development is that of the thousands of characteristics encoded on the DNA strand, all but one of them are "silenced." For example, in the particular cell which becomes a liver, only the "liver DNA" has an active voice, while the eye-color DNA is dormant. In the process of cloning, one adult cell (in the case of Dolly, a cell from a mammary gland) is stimulated to such a point whereby all the thousands of characteristics are "re-activated" - thereby enabling a new creature to fully develop from this single cell. (This was the basis of Woody Allen's 1973 film, "Sleeper," where a world leader suffers a tragic accident and is reconstructed from his nose.) There are 3 main steps to cloning: 1) All the DNA is removed from a female's egg. The egg is now a hollow shell, contributing no genetic characteristics of the egg-donor. 2) Another adult cell, which has been "re-activated," is now fused into this hollow egg, by means of electric current. This is called "nuclear transplantation." 3) The now-fertilized egg is implanted into the womb of another female (the surrogate mother). The fetus, which develops through a normal gestation period, is genetically identical to the original cell-donor.

Cloning:
The process of creating a genetically identical copy DNA Clones Just over 100 years ago, biologists were on a roll: Equipped with excel-lent, highresolution compound microscopes, and a few histochemical techniques, they were having tremendous success with the identification-tion and treatment of many diseases that were caused by bacteria. Robert Koch, a German country doctor, proved that anthrax, a fatal disease of cattle and humans, is caused by a bacterium, which he named Bacillus anthracis. Shortly after Kochs discovery, the great French chemist Louis Pasteur developed a vaccine, the first ever produced to treat anthrax. He quickly followed it up with another vaccine to treat rabies, a disease that was very common in Europe at the time. Paul Ehrlich, who worked in Kochs laboratory, produced a dramatically effective drug, which he called a magic bullet, to treat syphilis. The combined efforts of Koch, Pasteur, and Ehrlich led to treatments for tuberculosis, diphtheria, typhoid fever, and cholera. The knowledge they gained and the techniques they developed transformed the field of medicine from a confused, superstitious muddle into a highly efficient discipline for fighting and controlling infectious diseases. Cell Clones Any cell that reproduces asexually is a clone. Bacteria reproduce by binary fission, which is an asexual mode of reproduction. Conse- quently, bacteria, and indeed all prokaryotes, represent the largest group of clonal organisms on Earth. The fact that bacteria reproduce by cloning themselves seems to be at odds with their amazing adaptability and, in the case of pathogenic bacteria, their ability to develop resist- ance to antibiotics. A strain of Staphylococcus aureus has recently been isolated from a hospital ward that is resistant to all known clinical antibiotics. How can bacteria develop new characteristics such as this when the daughter cells are always genetically identical to the parent cells? How

does a daughter cell suddenly become resistant to an antibi- otic that would have killed its parent? The answer to these questions lies with the diversity of the prokaryotes, their short 20-minute generation time, and the existence of plasmids. Animal Clones Most of the animals that clone themselves are either marine or fresh- water invertebrates. This is not to imply that these animals never have sex, since most of them do, but clonal reproduction has become a cru- cial part of their survival strategy. All these animals are either sessile (attached to rocks or ground) or move very slowly.

Invertebrate clonal reproduction. Sea cucumbers, sea stars, and planaria can all reproduce asexually by fission or fragmentation. The hydra can clone itself by forming a bud that eventually separates and grows to adult size.

Sea cucumbers look something like the vegetable they are named after, but ones that are covered in rows of sharks teeth and crowned with a mane of wormlike tentacles. This animal forages on the seabed, where it feeds on detritus; it is affectionately referred to as an ocean- going vacuum cleaner. Being a slow-moving creature, it has to cope with fast-moving predators, a fact that has shaped both the sexual and asexual behavior of this animal. Sexually, sea cucumbers reproduce through a process called broadcast fertilization, whereby the males and females simply release their gametes into the water, where fertilizationand development of the embryos and larvae take place.

Cloning of a gene The production of many identical copies of a gene is called gene cloning.
A very large quantity of a gene is produced by recombinant DNA technology. The polymerase chain reaction (PCR) produces a lesser number of copies within a laboratory test tube.
Recombinant DNA Technology
The DNA which contains DNA from two different sources is called Recombinant DNA and the technology for the formation of recombinant DNA is called DNA technology.
Following material is required for producing recombinant DNA:

1. 2. 3.

Gene of interest: The genes are to be cloned.

Molecular scissors: These are used to cut out the gene of interest. Molecular carrier or vector: The genes of interest can be placed on it for transport. Expression system: The gene of interest with the vector is introduced into an expression
system. Thus a specific product is made.

How to get a gene?

There are three possible ways to get the gene of interest. 1. Isolation of gene from the chromosome: Genes can be isolated from the chromosomes by cutting the chromosomes on the flanking sites of the gene. Special enzymes restriction endonucleases are used to cut the genes.
2. Chemical synthesis of genes:
Small genes can be synthesized in the laboratory. 3. Making gene from mRNA: It is a very common method of getting the gene. In this case, gene is synthesized in the laboratory from messenger RNA by reverse transcriptase enzyme. This DNA molecule is called complementary DNA (coda).

Recombinant DNA

The plasmids are cut with the enzyme for preparation of a recombinant DNA. It is the same enzyme which was used for isolation of the gene of interest. The gene of interest (insulin) is then joined with the sticky ends of plasmids with the help of another special enzyme known as DNA ligase. This enzyme seals the foreign piece of DNA into the vector. Now the two different pieces of DNA have been joined together. It is now known as recombinant DNA or chimaeric DNA.

Expression of the Recombinant DNA

A clone can be a large number of molecules (i.e. cloned genes) or cell (i.e. cloned bacteria) or organisms that are identical to an original specimen.

1. The bacterial cells are treated with calcium chloride. It makes the bacterial
membrane more permeable. Now the bacterial celrs take up recombinant plasmid. 2. These bacteria reproduce and bacterial clones are formed. Each new cell contains at least one plasmid. Therefore, each clone of bacteria contains the gene of interest, 3 . The clone bacteria express themselves and make a product. 4 . The protein product can be separated from the done bacteria. The cloned gene can be isolated from this bacterial clone for further analysis.

Transgenic Plants
The plants with foreign DNA are called transgenic plants. Following techniques are developed for introducing foreign genes into plants:

1. Genes are inserted into immature plant embryos by a special technique. This embryo develops to form new transgenic plant. 2. Genes are inserted into protoplast of plant cells. The plant cells whose cell wall is removed are called protoplasts. The protoplasts are suspended in a liquid containing foreign DNA. Electric current are passed through this liquid. The electric current makes tiny, self-sealing holes in .the. plasma membrane. The genetic material enters into protoplast through these holes. Then a protoplast will develop into a complete plant.

Uses of transgenic plants:

There following uses of transgenic

Pest and herb resistance:

Foreign genes are transferred to cotton, corn and potato. The cell of these transgenic plants produces an insect toxin. So these plants become resistant to pests. Similarly, soybeans have been made resistant to a common herbicide .Some corn and cotton plants are both pest and herbicide resistant.

Transgenic crops:
In 1999, these transgenic crops were planted on more than 70 million acres worldwide. Their acreage (total acre) is expected to become triple in about five years. Improvements is still going on in these varieties. So that protein or starch content and modified oil or amino acid composition are increased in these varieties

The structure of stomata can be changed to increase carbon dioxide intake or cut down on water loss. Increasing the efficiency of the enzyme RuBP carboxylase. It captures CO2 in most plants. A team of Japanese scientists are attempting to introduce the C4 cycle into the rice. The C4 cycle uses a different method of capturing CO2 So C4 cycle reduces the inefficiency of RuBP carboxylase. These are multi gene modification. These modifications will require a thorough re-engineering of plant cells.

Single gene transfers:

So far, only single gene transfers have been done. Various products are produced by single gene transfers.
A weed called mouse-eared cress has been genetically engineered. It p r o d u c e s a b i o d e g r a d a b l e p l a s t i c ( p o l yh y d ro x y - bu t y r a t e ) i n c e l l granules.

5. Synthesis of human products:

Plants are being engineered to produce human hormones, clotting factors, and antibodies in their seeds. An antibody made by corn can deliver radio isotopes to tumor cells. Another antibody made by soybeans is used for the treatment of genital herpes.

The antibodies produced by plant are inexpensive These antibodies are not contaminated with pathogens The pathogens can infect people. Clinical trials of these human products have begun.

Transgenic Animals
Two techniques have been developed to insert genes into the egg s of animals. 1. Microinjection of genes: There can be micro injection of foreign genes into eggs by hand. 2. Vortex mixing: The eggs are placed in an agitator with DNA and silicon - carbide needles The needles make tiny holes through which the DNA can enter. These eggs are fertilized and produce transgenic offspring, Application: The technique of producing transgenic animals has following applications: 1. Higher growth rate of animals Transgenic: The genes for bovine (cattle) growth hormone are injected by this technique into many types of animal eggs. Thus many larger fishes, cows, pigs, rabbits and sheep are produced by this procedure. There is much concern that genetically engineered fishes will upset or destroy the natural ecosystems Thus these engineered fishes are kept in separate ponds. So that these fishes may not escape and mix with wild fishes.

2. Gene pharming: The use of transgenic farm animals to produce pharmaceuticals is called gene farming. A number of firms are using this technique. In this technique, the genes of therapeutic (antibodies) and diagnostic proteins are incorporated into the animal's DNA. Thus these proteins appear in the animal's milk. The scientists are trying to produce drugs for the treatment of cystic fibrosis, cancer, blood diseases and other disorders. Antithrombin III is produced by a herd of goats. It prevents the blood clotting during surgery. The clinical trials of antithrombin III have begun. Procedure of producing transgenic mammals for gene farming:
(i). DNA containing the gene of interest is injected into donor eggs. (ii). The egg is fertilized in vitro (in test tube) and zygote is formed. (iii). The zygotes are placed in host females. The offsprings are developed. (iv). Female offspring become mature. The product is secreted in the milk. (v). The scientists of United States Department of Agriculture have produced

genetically engineered mice. This mice is used to produce human growth hormone in their urine instead of milk. They will use the same technique on larger animals. Urine is preferred over milk for selection of biotechnology product due to these reasons: All animals in a herd urinate. While only females produce milk. Animals start urinating at birth. While females don't produce milk until maturity. Thus it is easier to extract proteins from urine than farm milk.

Cloning of Transgenic Animals

The production of the genetically identical copies of the organisms or cells by asexual reproduction is called cloning. The animals are genetically engineered to produce a

biotechnology product. Now identical copies of these animals are obtained by asexual reproduction through cloning. Cloning requires only the genes of one animal. So cloning is a form of asexual reproduction. It was earlier believed that adult vertebrate animals could not be cloned. Each cell contains a copy of all the genes. But certain genes are turned off in mature specialized cells. Different genes are expressed in muscle cells for contraction. While different genes are expressed in nerves cells for conduction a nerve impulses. Similarly different cells are expressed in gland cell for secretion. All genes of an adult cell should be turned on for cloning of normal developmert. It was earlier believed that cloning is impossible. In 1997, scientists at the Roslin Institute in Scotland produced a cloned sheep called Dolly . Since then calves and goats have been cloned.

1: Biology Trend Series 2nd year 2: http://www.scribd.com/doc/7209295/Introductionof-Cloning#archive 3:http://www.scienceclarified.com/scitech/Genetics/Ge netic-Engineering.html