What is cloning?

Types of Cloning

There are three different definitions of a clone. One is a group of genetically identical cells descended from a single common ancestor. This type of clone is often made by plant cell tissue culture in which a whole line of cells is made from a single cell ancestor. A second type of clone is a gene clone, or recombinant DNA clone, in which copies of a DNA sequence are made by genetic engineering. A third type of clone is an organism that is descended asexually from a single ancestor. A much-celebrated example of an organismal clone is the sheep Dolly (1997-2003), produced by placing the nucleus of a cell from a ewe’s udder, with its genetic material (DNA), into an unfertilized egg from which the nucleus had been removed.

DNA Cloning

DNA is cloned to obtain specific pieces of DNA that are free from other DNA fragments. Clones of specific pieces of DNA are important for basic research. DNA is made up of four different compounds known as nucleotide bases. Once a piece of DNA is cloned, the specific DNA bases can be identified. This is called sequencing. Once this specific pattern of DNA sequencing is accomplished, the DNA is said to be “sequenced,” revealing the genetic code detailed by the nucleotide bases. This valuable information helps answer the following questions and can be used in a variety of ways. Where does the gene begin and end? What type of control regions does the gene have? Cloned DNAs can be used as hybridization probes, where sequences that are complementary to the cloned DNA can be detected. Such DNA hybridization is useful to detect similarities between genes from different organisms, to detect the presence of specific disease genes, and to determine in what tissues that gene is expressed. The gene is expressed when a messenger RNA (mRNA) is made from the gene and the mRNA is translated into a protein product. A DNA clone is also used to produce the protein product for which that gene codes. When a clone is expressed, the protein made by that gene can be studied or an antibody against that protein can be made. An antibody is used to show in which tissues of an organism that protein is found. Also, a DNA clone may be expressed because the gene codes for a useful product. This is a way to obtain large amounts of the specific protein.

Products of Recombinant DNA Technology

Recombinant DNA technology has produced clones put to use for a wide variety of human purposes. For example, rennin and chymosin are used in cheese making. One of the most important applications, however, is in medicine. Numerous recombinant DNA products are useful in treating human diseases, including the production of human insulin (Humalin) for diabetics. Other human pharmaceuticals produced by gene cloning include clotting factor VIII to treat hemophilia A, clotting factor IX to treat hemophilia B, human growth hormone, erythropoietin to treat certain anemias, interferon
to treat certain cancers and hepatitis, tissue plasminogen activator to dissolve blood clots after a heart attack or stroke, prolastin to treat genetic emphysemas, thrombate III to correct a genetic antithrombin III deficiency, and parathyroid hormone. The advantages of the cloned products are their high purity, greater consistency from batch to batch, and the steady supply they offer.

How to Clone DNA

DNA is cloned by first isolating it from its organism. Vector DNA must also be isolated from bacteria. (A vector is a plasmid or virus into which DNA is inserted.) Both the DNA to be cloned and the vector DNA are cut with a restriction enzyme that makes sequence-specific cuts in the DNAs. The ends of DNA molecules cut with restriction enzymes are then joined together with an enzyme called ligase. In this way the DNA to be cloned is inserted into the vector. These recombinant DNA molecules (vector plus random pieces of the DNA to be cloned) are then introduced into a host, such as bacteria or yeast, where the vector can replicate. The recombinant molecules are analyzed to find the ones that contain the cloned DNA of interest.

Regulation of DNA Cloning

In the 1970s the tools to permit cloning of specific pieces of DNA were developed. There was great concern among scientists about the potential hazards of some combinations of DNA from different sources. Concerns included creating new bacterial plasmids with new drug resistances and putting DNA from cancer-causing viruses into plasmids. In February, 1975, scientists met at a conference center in Asilomar, California, to discuss the need to regulate recombinant DNA research. The result of this conference was the formation of the Recombinant DNA Molecule Program Advisory Committee at the National Institutes of Health, and guidelines for recombinant DNA work were established.

Genetically Modified Organisms

Numerous cloned genes have been introduced into different organisms to produce genetically modified organisms (GMOs). Genes for resistance to herbicides and insects have been introduced into soybean, corn, cotton, and canola, and these genetically engineered plants are in cultivation in fields in the United States and other countries. Fish and fruit and nut trees that mature more rapidly have been created by genetic engineering. Edible vaccines have been made—for example, a vaccine for hepatitis B in bananas. A tomato called the Flavr Savr is genetically engineered to delay softening. Plants that aid in bioremediation by taking up heavy metals such as cadmium and lead are possible.

Concerns about genetically modified organisms include safety issues—for example, concerns that foreign genes introduced into food plants may contain allergens and that the antibiotic resistance markers used in creating the GMOs might be transferred to other organisms. There are concerns about the environmental impact of GMOs; for example, if these foreign genes are transferred to other plants by unintended crossing of a GMO with a weed plant, weeds may become difficult or impossible to eradicate and jeopardize crop growth. There is a concern about the use of genetically modified organisms as food. There is a concern about loss of biodiversity if only one, genetically modified, variety of a crop plant is cultivated. There are also ethical concerns surrounding whether certain GMOs might be made available only in rich countries, and there are concerns about careful labeling of GMOs so that consumers will be aware when they are using products from GMOs. All of these questions remain in flux as the marketing of GMOs proceeds.

According to the International Service for the Acquisition of Agri-Biotech Applications (ISAAA), genetically modified (GM) crops were planted in twenty-seven countries by approximately 18 million farmers worldwide. Over 60 percent of the world's population live in the twenty-seven countries that are planting GM crops. In 2006, US government statistics showed that 87 percent of the global genetically modified crops were grown in developed countries. By 2013, however, ISAAA reported that Latin American, Asian, and African farmers grew 54 percent of the global GM crops compared to the 46 percent grown in developed countries worldwide. Corn, soybeans, cotton, alfalfa, and canola were the major crops, often modified for insect resistance. Rice has been genetically enhanced for more iron and vitamins to alleviate malnutrition in Asia. Other plants have been modified to survive weather variances.

Genetically modified organisms may soon include cows resistant to mad cow disease and nut and fruit trees that yield bounties faster. Plants producing new plastics and fish that grow faster are potential genetically modified organisms. It is expected that the world will see huge increases in genetically modified organisms as researchers gain more access to genomic resources.

Organismal Cloning

A goal of organismal cloning is to develop ways of efficiently altering animals genetically in order to reproduce certain animals that are economically valuable. Animals have been altered by the introduction of specific genes, such as human proteins that will create drug-producing animals. Some genes have been inactivated in organisms to create animal models of human diseases. For example, “knockout mice” are used as models for diabetes research. Another goal is to conduct research that might lead to the development of human organs for transplant produced from single cells. Similarly, animals might be genetically engineered to make their organs better suited for transplantation to humans. Finally, the cloning of a human might be a solution to human infertility.

Are Organismal Clones Normal?

There is, however, a concern about the health of cloned animals. First of all, when inserting a new nucleus into an egg from which the nucleus has been removed, and then implanting such eggs into surrogate mothers, only very few of the eggs develop properly. There are suggestions of other abnormalities in cloned animals that might be due to the cloning process. The first vertebrate to be successfully cloned, the sheep Dolly, developed first arthritis and then a lung disease when six years old; although neither condition was unusual in sheep, both appeared years earlier than normal, and Dolly was euthanized. Was she genetically older than her chronological age?

Stem Cells

Stem cells are unspecialized cells that are able to divide continuously and, with the proper conditions, be induced to give rise to specialized cell types. In the developing embryo they give rise to the hundreds of types of specialized cells that make up the adult body. Embryonic stem cells can be isolated from three- to five-day-old embryos. Some tissues in the adult, such as bone marrow, brain, and muscle, contain adult stem cells that can give rise to cell types of the tissue in which they reside.

A goal of research on stem cells is to learn how stem cells become specialized cells. Human stem cells could be used to generate tissues or organs for transplantation and to generate specific cells to replace those damaged as a result of spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, rheumatoid arthritis, and other conditions.

A 2012 study demonstrated that human corneas can be cultivated from stem cells and grown onto the damaged corneas of individuals who are at risk for blindness. This new technology does away with the need for cornea donors. Prior to this discovery, corneas could only be replaced by a donated cornea, which historically had been in short supply. (The cornea is the outermost portion of the eye and provides protection along with 70 percent of the eye’s focusing power.)

Regulation of Organismal Cloning

Until the cloning of the sheep Dolly in 1997, it was thought that adult specialized cells could not be made to revert to nonspecialized cells that can give rise to any type of cell. However, Dolly was created from a specialized adult cell from a ewe’s udder. After the publicity about Dolly, US president Bill Clinton asked the National Bioethics Advisory Commission to form recommendations about the ethical, religious, and legal implications of human cloning. In June, 1997, that commission concluded that attempts to clone humans are “morally unacceptable” for safety and ethical reasons. There was a moratorium on using federal funds for human cloning. In January, 1998, the US Food and Drug Administration (FDA) declared that it had the authority to regulate human cloning and that any human cloning must have FDA approval.

While there is general agreement in the United States and in many other countries that reproductive human cloning should be banned because of ethical and safety concerns, there is ongoing debate about whether or not to allow therapeutic cloning to treat human disease or research cloning to study how stem cells develop. The Human Cloning Prohibition Act of 2001 to ban both reproductive and therapeutic cloning passed in the US House of Representatives, but the Senate did not support the ban. The ban was again considered by the lawmakers in 2002. In the meantime, individual states such as California and New Jersey have passed bills that approve of embryonic stem cell research with the goal of leading to treatments for diseases such as Parkinson’s, diabetes, and Alzheimer’s. The research is controversial because embryos must be destroyed to obtain the stem cells, and some groups believe that constitutes taking a human life. The embryos used are generally extra embryos left over from in vitro fertilizations. In December 2002 and January 2003, a company called Clonaid announced the births of several babies it claimed were the result of human cloning but then failed to produce any scientific evidence that the babies were clones. In February, 2003, the US Congress considered a ban on both reproductive and therapeutic cloning. In late February, the House passed the Human Prohibition Cloning Act of 2003, banning the cloning of human beings but allowing limited research on some existing stem cell lines.

In May, 2008, President George W. Bush signed into law the Genetic Information Nondiscrimination Act (GINA). GINA prohibits US employers and insurance companies from discriminating on the basis of genetic test information, and insurance companies may not discriminate with reduced coverage or increased pricing based on information derived from genetic testing. Employers are prohibited from making adverse employment decisions based on an individual’s genetic code. Under GINA law, insurers and employers may not demand or request a genetic test.

GINA protections are meant to encourage increased genetic testing without the fear of job loss or insurance complications. It is hoped that more genetic testing will enable researchers to devise therapies for a wide range of hereditary diseases. Genetic testing may also enable earlier treatments with better outcomes and decreased health care costs.

Executive order 13505, “Removing Barriers to Responsible Scientific Research Involving Human Stem Cells,” was issued by President Barack Obama in March, 2009. This executive order requires the Health and Human Services secretary and the National Institutes of Health (NIH) director to review and issue new NIH guidelines regarding scientific research and human stem cells.

The tension between scientific possibility, public policy, and societal values continues in the arena of cloning. Through therapeutic cloning there is great potential for the treatment of human diseases, but the ethical concerns about such procedures must be carefully considered as well.

Key terms



cloning vector

:

a plasmid or virus into which foreign DNA can be inserted to amplify the number of copies of the foreign DNA in the host cell or organism





DNA


:

dexoyribonucleic acid, a long-chain macromolecule, made of units called nucleotides and structured as a double helix joined by weak hydrogen bonds, that forms genetic material for most organisms




DNA hybridization

:

formation of a double-stranded nucleic acid molecule from single-stranded nucleic acid molecules that have complementary base sequences




ligase

:

an enzyme that joins recombinant DNA molecules together




plasmid

:

a DNA molecule that replicates independently of chromosomes





recombinant DNA technology


:

methods used to splice a DNA fragment from one organism into DNA from another organism and then clone the new recombinant DNA molecule




reproductive cloning

:

cloning to produce individual organisms




restriction enzyme

:

a protein (an enzyme) that recognizes a specific nucleotide sequence in a piece of DNA and causes a sequence-specific cleavage of the DNA





stem cells


:

cells that are able to divide indefinitely in culture and to give rise to specialized cells




therapeutic cloning

:

cloning to produce a treatment for a disease





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