Full project on clonning

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The possibility of human cloning, raised when Scottish scientists at Roslin Institute created the much-celebrated sheep "Dolly" (Nature 385, 810-13, 1997), aroused worldwide interest and concern because of its scientific and ethical implications. The feat, cited by Science magazine as the breakthrough of 1997, also generated uncertainty over the meaning of "cloning" --an umbrella term traditionally used by scientists to describe different processes for duplicating biological material


What is Cloning?

Cloning is the creation of an organism that is an exact genetic copy of another. This means that every single bit of DNA is the same between the two!

You might not believe it, but there are human clones among us right now. They weren't made in a lab, though: they're identical twins, created naturally. Below, we'll see how natural identical twins relate to modern cloning technologies.

How is cloning done?

You may have first heard of cloning when Dolly the Sheep showed up on the scene in 1997. Cloning technologies have been around for much longer than Dolly, though.

How does one go about making an exact genetic copy of an organism? There are a couple of ways to do this: artificial embryo twinning and somatic cell nuclear transfer. How do these processes differ?
1. Artificial Embryo Twinning

Artificial embryo twinning is the relatively low-tech version of cloning. As the name suggests, this technology mimics the natural process of creating identical twins.
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In nature, twins occur just after fertilization of an egg cell by a sperm cell. In rare cases, when the resulting fertilized egg, called a zygote, tries to divide into a two-celled embryo, the two cells separate. Each cell continues dividing on its own, ultimately developing into a separate individual within the mother. Since the two cells came from the same zygote, the resulting individuals are genetically identical.

Artificial embryo twinning uses the same approach, but it occurs in a Petri dish instead of in the mother's body. This is accomplished by manually separating a very early embryo into individual cells, and then allowing each cell to divide and develop on its own. The resulting embryos are placed into a surrogate mother, where they are carried to term and delivered. Again, since all the embryos came from the same zygote, they are genetically identical.
2. Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer, (SCNT) uses a different approach than artificial embryo twinning, but it produces the same result: an exact clone, or genetic copy, of an individual. This was the method used to create Dolly the Sheep.

What does SCNT mean? Let's take it apart:

Somatic cell: A somatic cell is any cell in the body other than the two types of reproductive cells, sperm and egg. These are also called germ cells. In mammals, every somatic cell has two complete sets of chromosomes, whereas the germ cells only have one complete set.

Nuclear: The nucleus is like the cell's brain. It's an enclosed compartment that contains all the information that cells need to form an organism. This information comes in the form of DNA. It's the differences in our DNA that make each of us unique.

Transfer: Moving an object from one place to another.

To make Dolly, researchers isolated a somatic cell from an adult female sheep. Next, they transferred the nucleus from that cell to an egg cell from which the nucleus had been removed. After a couple of chemical tweaks, the egg cell, with its new nucleus, was behaving just like a freshly fertilized zygote. It developed into an embryo, which was implanted into a surrogate mother and carried to term.

The lamb, Dolly, was an exact genetic replica of the adult female sheep that donated the somatic cell nucleus to the egg. She was the first-ever mammal to be cloned from an adult somatic cell.
How does SCNT differ from the natural way of making an embryo?
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The fertilization of an egg by a sperm and the SCNT cloning method both result in the same thing: a dividing ball of cells, called an embryo. So what exactly is the difference between these methods?

An embryo is composed of cells that contain two complete sets of chromosomes. The difference between fertilization and SCNT lies in where those two sets originated.

In fertilization, the sperm and egg both contain one set of chromosomes. When the sperm and egg join, the resulting zygote ends up with two sets - one from the father (sperm) and one from the mother (egg).

In SCNT, the egg cell's single set of chromosomes is removed. It is replaced by the nucleus from a somatic cell, which already contains two complete sets of chromosomes. Therefore, in the resulting embryo, both sets of chromosomes come from the somatic cell.

Introduction

The possibility of human cloning, raised when Scottish scientists at Roslin Institute created the much-celebrated sheep "Dolly" (Nature 385, 810-13, 1997), aroused worldwide interest and concern because of its scientific and ethical implications. The feat, cited by Science magazine as the breakthrough of 1997, also generated uncertainty over the meaning of "cloning" --an umbrella term traditionally used by scientists to describe different processes for duplicating biological material.

What is cloning? Are there different types of cloning?

When the media report on cloning in the news, they are usually talking about only one type called reproductive cloning. There are different types of cloning however, and cloning technologies can be used for other purposes besides producing the genetic twin of another organism. A basic understanding of the different types of cloning is key to taking an informed stance on current public policy issues and making the best possible personal decisions. The following three types of cloning technologies will be discussed: (1) recombinant DNA technology or DNA cloning, (2) reproductive cloning, and (3) therapeutic cloning.


Recombinant DNA Technology or DNA Cloning

The terms "recombinant DNA technology," "DNA cloning," "molecular cloning,"or "gene cloning" all refer to the same process: the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid. The DNA of interest can then be propagated in a foreign host cell. This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today.

Scientists studying a particular gene often use bacterial plasmids to generate multiple copies of the same gene. Plasmids are self-replicating extra-chromosomal circular DNA molecules, distinct from the normal bacterial genome (see image to the right). Plasmids and other types of cloning vectors are used by Human Genome Project researchers to copy genes and other pieces of chromosomes to generate enough identical material for further study.

To "clone a gene," a DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes and then united with a plasmid that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA molecule." Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA. See a diagram depicting this process.

Plasmids can carry up to 20,000 bp of foreign DNA. Besides bacterial plasmids, some other cloning vectors include viruses, bacteria artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Cosmids are artificially constructed cloning vectors that carry up to 45 kb of foreign DNA and can be packaged in lambda phage particles for infection into E. coli cells. BACs utilize the naturally occurring F-factor plasmid found in E. coli to carry 100 to 300 kb DNA inserts. A YAC is a functional chromosome derived from yeast that can carry up to 1 MB of foreign DNA. Bacteria are most often used as the host cells for recombinant DNA molecules, but yeast and mammalian cells also are used.


Reproductive Cloning
Celebrity Sheep Has Died at Age 6

Dolly, the first mammal to be cloned from adult DNA, was put down by lethal injection Feb. 14, 2003. Prior to her death, Dolly had been suffering from lung cancer and crippling arthritis. Although most Finn Dorset sheep live to be 11 to 12 years of age, postmortem examination of Dolly seemed to indicate that, other than her cancer and arthritis, she appeared to be quite normal. The unnamed sheep from which Dolly was cloned had died several years prior to her creation. Dolly was a mother to six lambs, bred the old-fashioned way.

Image credit: Roslin Institute Image Library, http://www.roslin.ac.uk/imagelibrary/


Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly was created by reproductive cloning technology. In a process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed. The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth.

Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal. Only the clone's chromosomal or nuclear DNA is the same as the donor. Some of the clone's genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria, which are organelles that serve as power sources to the cell, contain their own short segments of DNA. Acquired mutations in mitochondrial DNA are believed to play an important role in the aging process.

Dolly's success is truly remarkable because it proved that the genetic material from a specialized adult cell, such as an udder cell programmed to express only those genes needed by udder cells, could be reprogrammed to generate an entire new organism. Before this demonstration, scientists believed that once a cell became specialized as a liver, heart, udder, bone, or any other type of cell, the change was permanent and other unneeded genes in the cell would become inactive. Some scientists believe that errors or incompleteness in the reprogramming process cause the high rates of death, deformity, and disability observed among animal clones.


Therapeutic Cloning

Therapeutic cloning, also called "embryo cloning," is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Stem cells are important to biomedical researchers because they can be used to generate virtually any type of specialized cell in the human body. Stem cells are extracted from the egg after it has divided for 5 days. The egg at this stage of development is called a blastocyst. The extraction process destroys the embryo, which raises a variety of ethical concerns. Many researchers hope that one day stem cells can be used to serve as replacement cells to treat heart disease, Alzheimer's, cancer, and other diseases.

In November 2001, scientists from Advanced Cell Technologies (ACT), a biotechnology company in Massachusetts, announced that they had cloned the first human embryos for the purpose of advancing therapeutic research. To do this, they collected eggs from women's ovaries and then removed the genetic material from these eggs with a needle less than 2/10,000th of an inch wide. A skin cell was inserted inside the enucleated egg to serve as a new nucleus. The egg began to divide after it was stimulated with a chemical called ionomycin. The results were limited in success. Although this process was carried out with eight eggs, only three began dividing, and only one was able to divide into six cells before stopping.


Celebrity Sheep Has Died at Age 6

Dolly, the first mammal to be cloned from adult DNA, was put down by lethal injection Feb. 14, 2003. Prior to her death, Dolly had been suffering from lung cancer and crippling arthritis. Although most Finn Dorset sheep live to be 11 to 12 years of age, postmortem examination of Dolly seemed to indicate that, other than her cancer and arthritis, she appeared to be quite normal. The unnamed sheep from which Dolly was cloned had died several years prior to her creation. Dolly was a mother to six lambs, bred the old-fashioned