Clones and Cloning
Cloning involves creating a population of genetically identical cells by asexual reproduction. A second definition involves creating an exact replica of a gene, or gene cloning.
Cloning is a natural process. Natural cloning occurs in unique ways. For example, identical twins start as a single fertilized cell, which then divides into two complete entities. Since ancient times pants have been known to propagate by asexual means. These genetically identical plants are clones. Cuttings of the leaves, stems, or roots clone some varieties.
Identical cell cloning is done by nuclear transfer. This idea is not new. In 1952 scientists cloned frogs with identical genetic materials. In the 1980s, researchers removed the nucleus from a body cell of a mouse embryo and put it into the fertilized egg of another mouse. Also, in the 1980s, animal research organizations began looking for ways to produce live calves by nuclear transfer from embryos. In 1987, Tracy, a transgenic sheep, was developed that secreted human proteins in her milk. The term transgenic is given to animals that have the genes of another. This transfer involves removing the DNA sequence that contains a gene and replacing it with a sequence of DNA. The term knockout is used to describe a gene removed or rendered inactive. Transgenic livestock and genetically modified pigs are a potential source of organs for transplantation to people. Also, animals, mostly mice, are engineered to simulate diseases like diabetes, arteriosclerosis, and Alzheimer's disease.
A major breakthrough in cloning came in 1995, when Scottish scientist Ian Wilmut (1944-) and colleagues at the Roslin Institute in Edinburgh produced live lambs, Megan and Morag, by nuclear transfer. The cells from early embryos had been cultured for months in the laboratory. The scientists considered the success due to their ability to induce quiescence in the cell. This state occurs when the basic function of the cell has shut down, making it suitable for nuclear transfer. Other experiments were done at the time to see if the nuclear transfer could only be done with embryo cells or could other cell types be used. Four lambs were born from embryo cells, three from fetal cells, and one from adult cells. The name of the lamb produced from adult cells was Dolly.
Dolly challenged a fundamental tenet of developmental biology. She was the first mammal cloned from the cell of an adult animal. Cells from a six-year-old ewe were cultured in a lab and then fused with unfertilized eggs with the genetic material removed. Twenty-nine eggs developed normally and were implanted in 13 ewes. Only one gave birth, to Dolly,148 days later.
Scientists continue to progress toward applications for cloning in medicine. In July 1997, Roslin announced Polly, the first transgenic lamb, was produced by nuclear transfer with human genes. The donor cell was a fetal fibroblast that had been transfected with a gene coding for human blood clotting factor IX. This factor is missing in persons with blood disorders like hemophilia and has great potential for assisting those with this condition. Human proteins like this have potential in the treatment of a variety of diseases.
Cloning and genetic modifications are promising in many areas. Nutriceuticals is a new branch of investigation for therapy with nutrition. For example, some individuals are allergic to cow's milk. Gene targeting would allow the cow's protein to be replaced by human proteins, which may make it more palatable. People with lactose intolerance could get the nutrition in milk minus the problem proteins. Transplantation of organs like heart and kidneys has resulted in a shortage of organs for transfer. Transgenic pigs, with an added human protein of inhibiting factors, could provide organs to alleviate the shortage.
The pharmaceutical possibilities of cloning show promising potential. In December 2000, the lab that cloned Dolly announced plans to engineer chickens that lay eggs containing cancer drugs. Animal models of disease are providing scientists with tools for studying the effect of drugs and other procedures.
Gene cloning is the production of exact copies of a particular gene using genetic engineering technology. The DNA containing the target gene is split into fragments using restriction enzymes. The fragments are put into cloning vectors, which are inserted into host cells. In the host cell the recombinant DNA replicates, and a colony of cells with the cloned gene develops. Using this technique, a clone bank or genomic library may be set up.
Chromosome walking is a strategy used to fill in gaps. Walking involves hybridizing a primer of known sequence to a clone from an unordered genomic library, then synthesizing a short complementary strand. The new strand is then sequenced, and the end used as a new primer for walking along the chromosome step by step. Chromosome walking can be used in cloning disease genes.
To isolate clones that are not contiguous, a technique known as chromosome jumping enables the scientist to skip a region between known points on the chromosome. This is done usually to bypass regions that are difficult or impossible to walk through.
Positional cloning allows researchers to map disease-linked genes to a specific chromosome. Using samples from families with a history of a disease, scientists use genetic mapping to track variation and look for markers related to the disease. Over fifty disease-linked genes have been located by positional cloning.
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