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Procedure of cloning
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WHAT IS CLONING ?

Cloning 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

HISTORY OF CLONING

1962 - John Gurdon claims to have cloned frogs from adult cells.
1963 - J.B.S. Haldane coins the term 'clone.'
1966 - Establishment of the complete genetic code.
1967 - Enzyme DNA ligase isolated.
1969 - Shapiero and Beckwith isolate the first gene.
1970 - First restriction enzyme isolated.
1972 - Paul Berg creates the first recombinant DNA molecules.
1973 - Cohen and Boyer create first recombinant DNA organisms.
1977 - Karl Illmensee claims to have created mice with only one parent.
1979 - Karl Illmensee makes claim to have cloned three mice.
1983 - Kary B. Mullis develops the polymerase chain reaction technique for rapid DNA synthesis.
1983 - Solter and McGrath fuse a mouse embryo cell with an egg without a nucleus, but fail to clone using their technique.
1984 - Steen Willadsen clones sheep from embryo cells
1985 - Steen Willadsen clones sheep from embryo cells. Steen Willadsen joins Grenad Genetics to commercially clone cattle.
1986 - Steen Willadsen clones cattle from differentiated cells.
1986 - First, Prather, and Eyestone clone a cow from embryo cells.
1990 - Human Genome Project begins
1996 - Dolly, the first animal cloned from adult cells, born.
1997 - President Bill Clinton proposes a five year moratorium on cloning.
1997 - Richard Seed announces his plans to clone a human.
1997 - Wilmut and Campbell create Polly, a cloned sheep with an inserted human gene.
1998 - Teruhiko Wakayama creates three generations of genetically identical cloned mice.
2000: Pigs and goats reported cloned from adult cells. Britain becomes the first country to grant a patent for cloned early-stage human embryos.

March 2000: Scientists announce first cloned pigs.

2001: An endangered Asian ox dies two days after the birth of an ordinary disease after it was cloned and gestated in the womb of a cow. Advanced Cell Technology of Worcester, Massachusetts, says it produced a six-cell cloned human embryo, in research aimed at harvesting stem cells.

2002: Rabbits and a kitten reported cloned from adult cells.

December 26, 2002: Clonaid claims to have produced the first human clone, a baby girl called Eve.




In 1962, biologist John Gurdon of Oxford University announced that he had used the nucleus of fully differentiated adult intestinal cells to clone South African frogs. Gurdon's results electrified the scientific community, but some scientists remained skeptical and began to find flaws in his work.

In 1963, the British biologist J.B.S. Haldane is credited to have coined the term "clone" in a speech entitled "Biological Possibilities for the Human Species of the Next Ten-Thousand Years." Even though many scientists had described, and even completed the cloning process by this time, the term "cloning" had never been used to describe such experiments.

In 1966, Marshall Niremberg, Heinrich Mathaei, and Severo Ochoa crack the genetic code. The cracking of the genetic code opened the door for the explosion of genetic engineering studies and achievements beginning in the late 1970's.

In 1967, the enzyme DNA ligase was isolated. DNA ligase binds together strands of DNA. Its discovery, with the isolation of the first restriction enzyme 1970, paved the way for the first recombinant DNA molecules to be created by Paul Berg in 1972. In the recombinant DNA process, ligase bonds the "sticky" ends of complimentary DNA strands previously cut by a restriction enzyme.

In 1969, James Shapiero of Harvard University, working with Johnathan Beckwith announce that they had isolated the first gene. The gene directed the digestion of sugar in a certain type of bacteria. Shapiero and Beckwith's discovery part of a wave of molecular biology discoveries directly following the 1966 cracking of the genetic code. The announcement also increased the public's concern about the growing power of molecular biologists.

In 1970, both Howard Temin and David Baltimore, working independently, isolated the first restriction enzyme. The restriction enzyme, called Reverse Transcriptase, cut DNA molecules at precise locations. This capability led to the future manipulation of DNA.

In 1972, Paul Berg of Stanford University created the first recombinant DNA molecules by combining the DNA of two different organisms.

In 1973, Stanley Cohen and Herbert Boyer created the first recombinant DNA organism using recombinant DNA techniques pioneered a year earlier by Paul Berg. Recombinant DNA, also called gene splicing, is a technique that allows scientists to manipulate the DNA of an organism.

In 1977, German developmental biologist Karl Illmensee, working with Peter Hoppe at Jackson Laboratory in Maine, created mice with only a single parent. They grew mice with only a father as well as mice with only a mother.

In 1979, One of the most surprising of modern genetics announcements was made, Karl Illmensee claimed to have cloned three mice in 1979. The announcement came at a time where a succession of failed cloning attempts were beginning to convince biologists that the cloning of a mammal was impossible.

In 1983, In what has been called by some the greatest achievement of modern molecular biology, Kary B. Mullis developed the polymerase chain reaction (PCR) in 1983. PCR allows the rapid synthesis of designated fragments of DNA. Using the technique, over one billion copies can be synthesized in a matter of hours.

In 1983 Davor Solter, working with David McGrath, attempted to clone mice using his own version of the nuclear transfer method. They wanted to use the cloning experiment to determine if DNA specializes as a cell specializes.

In 1984, Danish scientist Steen Willadsen succeeded in cloning a sheep from embryo cells. His work was the first verified cloning of a mammal using the method of nuclear transfer.

In 1985 Steen Willadsen, the first to clone a farm animal using the nuclear transfer method, joined Grenada Genetics, a bioengineering company. Willadsen used his cloning technique to duplicate the embryos of prize cattle. Grenada Genetics saw the profitability of the future cattle cloning industry. Top breed cattle embryos were highly desired by farmers, Willadsen's procedure mass produced identical copies of such embryos.

In 1986, while working at Grenada Genetics, Steen Willadsen cloned a cow using differentiated, one week old embryo cells. The work proved that the genetic information of a cell did not diminish as a cell specialized and that DNA could return to its original state. Willadsen never officially published his results, but the work was a strong influence in Ian Wilmut's decision to attempt to clone from adult cells, which he accomplished in the famous 1996 birth of "Dolly."

In 1986, Neal First, Randal Prather, and Willard Eyestone, working at the University of Wisconsin, cloned a cow from early embryo cells. Though the race to clone the first farm animal had already been won by Steen Willadsen in 1984, Prather, Eyestone, and First's project was undertaken roughly at the same time as Willadsen's.

In October of 1990, the National Institutes of Health officially began the Human Genome Project, a massive international collaborative effort to locate the 50,000 to 100,000 genes and sequence the estimated 3 billion nucleotides making up the entire human genome. By determining the complete genetic sequence, scientists hope to begin correlating human traits with certain genes. With this information, medical researchers have begun to determine the intricacies of human gene function, including the source of genetic disorders and diseases that have plagued medical researchers for years.

In July 1995, Ian Wilmut and Keith Campbell of the Roslin Institute in Scotland successfully cloned two sheep, named Megan and Morag, from differentiated embryo cells. The idea to clone sheep was arrived at by Ian Wilmut as an answer to a gene insertion project he was researching. At the time, time inserting genes into embryo cells was a difficult and tedious process. Few embryos survived the insertion of a gene, even fewer incorporated the gene into their genetic code, and even fewer organisms developed properly and used the gene in all of their cells.

On July 5, 1996, Dolly, the first organism ever to be cloned from adult cells, was born. Ian Wilmut and Keith Campbell, researchers at the Roslin Institute in Scotland created Dolly using a technique similar to that with which they created the first sheep from differentiated embryo cells in 1995.

On March 4, 1997, President Clinton, in response to the large scale human cloning ethics debate brought about by Ian Wilmut's announcement of the creation of Dolly, proposed a five year moratorium on federal and privately funded human cloning research. In addition to this proposal, Clinton asked the National Bioethics Advisory Commission to review the prospects of human cloning and determine if legal preventive actions should be taken.

On December 5, 1997, Harvard graduate Richard Seed announced that he planned to clone a human being before any federal laws could be enacted to ban the process. Seed's announcement added fuel to the raging ethical debate on human cloning that had been sparked by Ian Wilmut's creation of Dolly, the first clone obtained from adult cells.

In July 1997, building upon their success of the creation of Dolly, the first animal cloned from adult cells, Ian Wilmut and Keith Campbell created Polly, a Poll Dorset lamb cloned from skin cells grown in a lab and genetically altered to contain a human gene. Polly's birth signified the first step in the application of cloning technology to the production of a useful product. Most scientists believe that most beneficial application of cloning will come from the exact reproduction of animals genetically altered to produce human proteins or organs more easily accepted in transplants. Wilmut and Campbell's creation of Polly surprised the scientific community by how fast cloning technology was progressing. The cloning of genetically altered farm animals was not expected for another five years.

In July 1998, Ryuzo Yanagimachi, Toni Perry, and Teruhiko Wakayama of the University of Hawaii announced that they had cloned fifty mice from adult cells since October of 1997. The new cloning technique, which has proven to be more efficient than that performed by Ian Wilmut in his cloning of Dolly, was developed by postdoctoral student Wakayama in his spare time.

January 2000
Britain becomes the first country to grant a patent for cloned early-stage human embryos. Geron Corporation, which received the patent, says is has no intention of creating cloned humans.

March 2000
The group that created Dolly the sheep announces the first cloned pigs. Scientists hope that pigs could be genetically engineered for use in human organ transplants.

January 2001
An endangered Asian ox called a gaur dies two days after birth of an ordinary disease after it was cloned and gestated in the womb of a cow.
2002: Rabbits and a kitten reported cloned from adult cells.

December 26, 2002: Clonaid claims to have produced the first human clone, a baby girl called Eve.



Technology
Nuclear transfer involves transferring the nucleus from a diploid cell ( containing 30-40,000 genes and a full set of paired chromosomes) to an unfertilised egg cell from which the maternal nucleus has been removed. The technique involves several steps (see diagram below). The nucleus itself can be transferred or the intact cell can be injected into the oocyte. In the latter case, the oocyte and donor cell are normally fused and the 'reconstructed embryo' activated by a short electrical pulse. In sheep, the embryos are then cultured for 5-6 days and those that appear to be developing normally ( usually about 10%) are implanted into foster mothers.

Nuclear transfer is not a new technique. It was first used in 1952 to study early development in frogs and in the 1980's the technique was used to clone cattle and sheep using cells taken directly from early embryos. In 1995, Ian Wilmut, Keith Campbell and colleagues created live lambs- Megan and Morag - from embryo derived cells that had been cultured in the laboratory for several weeks. This was the first time live animals had been derived from cultured cells and their success opened up the possibility of introducing much more precise genetic modifications into farm animals.

In 1996, Roslin Institute and collaborators PPL Therapeutics created Dolly, the first animal cloned from a cell taken from an adult animal. The announcement of her birth in February 1997 started the current fascination in all things cloned. Until then, almost all biologists thought that the cells in our bodies were fixed in their roles: the creation of Dolly from a mammary gland cell of a six year old sheep showed this was not the case and the achievement was voted Science Breakthrough of the Year at the end of 1997.

for more details refer to roslin institute web site

INSTRUMENTATION IN CLONING
A compound microscope with a left and right hand micromanipulator is required in the cloning procedure. The microscope magnifies the specimen and the micromanipulators reduce the technician's hand movement to allow microsurgery. Each microtool is connected to Teflon tubing leading to a syringe system, allowing the technician to control the pressure at the tip of the microtools.

Fine, glass microtools are placed in the micromanipulator tool holders and directed into the petri dish over the microscope objective. The tool on the left, called the holding pipette, is used to hold the egg. The tool on the right, called the enucleation pipette, is used to remove the genetic material from the egg and then used to place the donor cell adjacent to the egg membrane. Using a fluid filled syringe system, the technician can hold on to the egg and manipulate the donor cell

Cell fusion device
A new cell fusion device specialized in embryonic cell fusion



for more details refer to lazaron.com