Animal Biotechnology

What is Biotechnology?

Biotechnology in one form or another has flourished since prehistoric times. When the first human beings realized that they could plant their own crops and breed their own animals, they learned to use biotechnology. The discovery that fruit juices fermented into wine, or that milk could be converted into cheese or yogurt, or that beer could be made by fermenting solutions of malt and hops began the study of biotechnology. When the first bakers found that they could make a soft, spongy bread rather than a firm, thin cracker, they were acting as fledgling biotechnologists. The first animal breeders, realizing that different physical traits could be either magnified or lost by mating appropriate pairs of animals, engaged in the manipulations of biotechnology.

What then is biotechnology?

The term brings to mind many different things. Some think of developing new types of animals. Others dream of almost unlimited sources of human therapeutic drugs. Still others envision the possibility of growing crops that are more nutritious and naturally pest-resistant to feed a rapidly growing world population. This question elicits almost as many first-thought responses as there are people to whom the question can be posed.
In its purest form, the term "biotechnology" refers to the use of living organisms or their products to modify human health and the human environment. Prehistoric biotechnologists did this as they used yeast cells to raise bread dough and to ferment alcoholic beverages, and bacterial cells to make cheeses and yogurts and as they bred their strong, productive animals to make even stronger and more productive offspring.
Throughout human history, we have learned a great deal about the different organisms that our ancestors used so effectively. The marked increase in our understanding of these organisms and their cell products gains us the ability to control the many functions of various cells and organisms. Using the techniques of gene splicing and recombinant DNA technology, we can now actually combine the genetic elements of two or more living cells. Functioning lengths of DNA can be taken from one organism and placed into the cells of another organism. As a result, for example, we can cause bacterial cells to produce human molecules. Cows can produce more milk for the same amount of feed. And we can synthesize therapeutic molecules that have never before existed.

Animal Biotechnology
Animal biotechnology is the application of scientific and engineering principles to the processing or production of materials by animals or aquatic species to provide goods and services (NRC 2003). Examples of animal biotechnology include generation of transgenic animals or transgenic fish (animals or fish with one or more genes introduced by human intervention), using gene knockout technology to generate animals in which a specific gene has been inactivated, production of nearly identical animals by somatic cell nuclear transfer (also referred to as clones), or production of infertile aquatic species.

The definition of animal biotechnology and its new biological products is a complex and still controversial issue. A transgenic organism is one that carries and expresses genetic information not normally found in that species of organism. This definition is somewhat literal and thus restrictive. Perhaps the term should be broadened to include the purposeful amplification, spread, or dissemination of a gene within a species at a rate much faster than would have occurred in the absence of artificial interventions. This broadened definition implies organisms that have been intentionally manipulated using modern biotechnological techniques to "design" both plants and animals ...

Transgenics
· Since the early 1980s, methods have been developed and refined to generate transgenic animals or transgenic aquatic species. For example, transgenic livestock and transgenic aquatic species have been generated with increased growth rates, enhanced lean muscle mass, enhanced resistance to disease or improved use of dietary phosphorous to lessen the environmental impacts of animal manure.
· Transgenic poultry, swine, goats, and cattle also have been produced that generate large quantities of human proteins in eggs, milk, blood, or urine, with the goal of using these products as human pharmaceuticals. Examples of human pharmaceutical proteins include enzymes, clotting factors, albumin, and antibodies.
· The major factor limiting widespread use of transgenic animals in agricultural production systems is the relatively inefficient rate (success rate less than 10 percent) of production of transgenic animals. CSREES has supported research projects to generate transgenic animals or transgenic aquatic species with enhanced production or health traits.

Gene Knockout Technology
· Animal biotechnology also can knock out or inactivate a specific gene. Knockout technology creates a possible source of replacement organs for humans.
· The process of transplanting cells, tissues, or organs from one species to another is referred to as “xenotransplantation.” Currently, the pig is the major animal being considered as a xenotransplant donor to humans. Unfortunately, pig cells and human cells are not immunologically compatible. Pig cells express a carbohydrate epitope (alpha1, 3 galactose) on their surface that is not normally found on human cells. Humans will generate antibodies to this epitope, which will result in acute rejection of the xenograft. Genetic engineering is used to knock out or inactivate the pig gene (alpha1, 3 galactosyl transferase) that attaches this carbohydrate epitope on pig cells.
· Other examples of knockout technology in animals include inactivation of the prion-related peptide (PRP) gene that may generate animals resistant to diseases associated with prions (bovine spongiform encephalopathy [BSE], Creutzfeldt-Jakob Disease [CJD], scrapie, etc.). Most of the funding for these types of projects is conducted by private companies or in academic laboratories supported by the National Institutes of Health. Research projects designed to provide basic information regarding mechanisms associated with gene knockout technology are supported by CSREES.

Somatic Cell Nuclear Transfer
· Another application of animal biotechnology is the use of somatic cell nuclear transfer to produce multiple copies of animals that are nearly identical copies of other animals (transgenic animals, genetically superior animals, or animals that produce high quantities of milk or have some other desirable trait, etc.). This process has been referred to as cloning.
· To date, somatic cell nuclear transfer has been used to clone cattle, sheep, pigs, goats, horses, mules, cats, rats, and mice. The technique involves culturing somatic cells from an appropriate tissue (fibroblasts) from the animal to be cloned. Nuclei from the cultured somatic cells are then microinjected into an enucleated oocyte obtained from another individual of the same or a closely related species.
· Through a process that is not yet understood, the nucleus from the somatic cell is reprogrammed to a pattern of gene expression suitable for directing normal development of the embryo.
· After further culture and development in vitro, the embryos are transferred to a recipient female and ultimately will result in the birth of live offspring. The success rate for propagating animals by nuclear transfer is often less than 10 percent and depends on many factors, including the species, source of the recipient ova, cell type of the donor nuclei, treatment of donor cells prior to nuclear transfer, the techniques employed for nuclear transfer, etc.

Production of Infertile Aquatic Species.

· In aquaculture production systems, some species are not indigenous to a given area and can pose an ecological risk to native species should the foreign species escape confinement and enter the natural ecosystem.
· Generation of large populations of sterile fish or mollusks is one potential solution to this problem. Techniques have been developed to alter the chromosome complement to render individual fish and mollusks infertile. For example, triploid individuals (with three, instead of two, sets of chromosomes) have been generated by using various procedures to interfere with the final step in meiosis (extrusion of the second polar body). Timed application of high or low temperatures, various chemicals, or high hydrostatic pressure to newly fertilized eggs has been effective in producing triploid individuals.
· At a later time, the first cell division of the zygote can be suppressed to produce a fertile tetraploid individual (four sets of chromosomes). Tetraploids can then be mated with normal diploids to produce large numbers of infertile triploids. Unfortunately, in a commercial production system, it is often difficult to obtain sterilization of 100 percent of the individuals; thus, alternative methods are needed to ensure reproductive confinement of transgenic fish.
· Another technique that is being developed for finfish is to farm monosex fish stocks. Monosex populations can be produced by gender reversal and progeny testing to identify XX males for producing all female stocks or YY males for producing all male stocks. CSREES has supported research projects to alter the chromosome content or produce monosex populations of genetically engineered fish or mollusks.

Uncertainties, safety issues and potential risks of animal Biotechnology
1. For example, concerns have been raised regarding: the use of unnecessary genes in constructs used to generate transgenic animals, the use of vectors with the potential to be transferred or to otherwise contribute sequences to other organisms, the potential effects of genetically modified animals on the environment, the effects of the biotechnology on the welfare of the animal, and potential human health and food safety concerns for meat or animal products derived from animal biotechnology.
2. Before animal biotechnology will be used widely by animal agriculture production systems, additional research will be needed to determine if the benefits of animal biotechnology outweigh these potential risks. The USDA Biotechnology Risk Assessment Grants program supports environmental risk assessment research projects on genetically engineered animals. In addition, the NRI Animal Protection program supports research projects to determine the effects of genetic modification on the health and well-being of the animal.

Advances in animal biotechnology have been facilitated by recent progress in sequencing and analyzing animal genomes, identification of molecular markers (microsatellites, expressed sequence tags [ESTs], quantitative trait loci [QTLs], etc.) and a better understanding of the mechanisms that regulate gene expression.
For more information on these topics and projects supported by CSREES in this area, see Animal Breeding, Genetics, and Genomics.

In Canada, the animal biotechnology sector, which includes research and development activities and the resultant animals and their products, is subject to the same rigorous health and safety regulations that apply to conventional animals and their derived products. These regulatory controls include the Health of Animals Act and Regulations, the Food and Drugs Act and Regulations, the Meat Inspection Act and Regulations, and the Feeds Act and Regulations, administered by the Canadian Food Inspection Agency (CFIA) and Health Canada. In addition, animals and their derived products produced through biotechnology are considered as "novel" or "new", triggering additional regulatory controls depending on the intended use of the product and/or its release into the environment.
Currently, animal biotechnology research is permitted in Canada, including research on livestock animals; however the animals must be housed in contained facilities to prevent release from the facility of the animal, its genetic material in living cells, or any material which might be associated with toxicity. To date, no animals produced using biotechnology have been approved for release into the Canadian environment, or into the food or feed chain.
The term "animal biotechnology" is an extension of the definition of biotechnology. This term may include, but is not limited to, the following categories of animals:
Genetically engineered or modified animals in which genetic material has been added, deleted, silenced or altered to influence expression of genes and traits.
Clones of animals derived by nuclear transfer from embryonic and somatic cells.
Chimeric animals that have received transplanted cells from another animal.
Interspecies hybrids produced by any methods employing biotechnology.
Animals derived by in vitro cultivation such as maturation or manipulation of embryos.

Health Canada considers novel foods, including animals produced through biotechnology, to be subject to the regulations in Division 28, Part B, of the Food and Drug Regulations. Therefore, developers producing animals through biotechnology must not introduce the products or by-products of these animals or their progeny into the human food supply in Canada, unless they have been subject to a pre-market safety assessment which is required for novel foods. More information on novel foods is posted on Health Canada's website.
The CFIA also considers novel feeds, including ingredients from animals produced through biotechnology, to be subject to assessment before any derived products and by-products can be released in the feed chain. More information regarding the use of ingredients derived from animal biotechnology in animal feeds can be found at the CFIA's website.
Animals produced though biotechnology and their progeny are also considered to be "new substances" under the Canadian Environmental Protection Act 1999 and must meet the Environment Canada notification requirements under the New Substances Notification Regulations. More information regarding the requirements for notification of new substances is posted on Environment Canada's website.
Information we provide for the general public includes:
Livestock and Animal Products Derived Through Modern Biotechnology: Roles and Responsibilities of the Government of Canada
Fish Products Derived Through Modern Biotechnology: Roles and Responsibilities of the Government of Canada
Information we provide to the livestock sector and scientific community includes:
Government of Canada's comments on the USFDA's document "Animal Cloning: a Draft Risk Assessment"
Guidelines
Animal Health Risk Analysis Framework for Biotechnology-Derived Animals
Notification Guidelines for the Environmental Assessment of the Use of Animal Biotechnology in Livestock
Summary Reports from Consultations
2004 - CFIA Consultation on Animal Biotechnology
2003 - CFIA Animal Biotechnology Focus Group Meeting
1998 - Development of a Regulatory Framework for Animal Biotechnology : Copies of presentations made during this meeting are available upon request.

Biotechnological Approaches To Vaccine productionIntroduction

At present, the majority of veterinary vaccines are produced by conventional methods similar to those implemented by Jenner or Pasteur. These include live, attenuated vaccines and killed or inactivated vaccines. Both of these types of vaccines have proven to be effective particularly in reducing the clinical manifestation following exposure to virulent filed strains of the pathogens.
One of the important impediments in the case of live vaccines is to ensure that the organism is attenuated sufficiently not to cause the disease, but still replicate to a sufficient level to induce an appropriate immune response. However, only a limited number of viral disease can be prevented by live attenuated viral vaccines state and most DNA-containing viruses have the potential to establish persistent (or latent) infection. New viral strains may arise by recombination of the vaccine virus with other viral strains in animal populations; pregnant animals or their offspring may be adversely affected by the vaccine strain.

Animal Biotechnology

Animal biotechnology is the application of scientific and engineering principles to the processing or production of materials by animals or aquatic species to provide goods and services (NRC 2003). Examples of animal biotechnology include generation of transgenic animals or transgenic fish (animals or fish with one or more genes introduced by human intervention), using gene knockout technology to generate animals in which a specific gene has been inactivated, production of nearly identical animals by somatic cell nuclear transfer (also referred to as clones), or production of infertile aquatic species.

Animals Overview

Animal agriculture is a significant portion of U.S. agriculture. Related research, education and extension activities are continually changing to address new challenges and opportunities brought about by rapidly advancing technologies, evolving consumer demand, and the need to make positive contributions to environmental, human, and animal health. Efficient, science-based animal agriculture translates into affordable and high-quality food for the consumer. NIFA and its land-grant university partners collaborate with industry and other interested parties to develop and disseminate knowledge and methods to improve agriculturally relevant animal systems.

Drug addiction

What is Drug Addiction?

Drug addiction is a complex brain disease. It is characterized by compulsive, at times uncontrollable, drug craving, seeking, and use that persist even in the face of extremely negative consequences.Drug seeking becomes compulsive, in large part as a result of the effects of prolonged drug use on brain functioning and, thus, on behavior. For many people, drug addiction becomes chronic, with relapses possible even after long periods of abstinence.

Symptoms and Diagnosis of Alcoholism, Addiction

Many times, determining whether someone is an actual alcoholic or not is really not all that important. If someone's drinking and drugging has advanced to the point to where it has become a problem for them and those around them, then it is a problem, period. If the person has tried to quit -- swore to themselves and others "never again!" -- and simply cannot stop, then chances are they have become dependent or addicted.

What Is Alcoholism?

Alcohol problems occur at different levels of severity, from mild and annoying to life-threatening. Although alcohol dependence (alcoholism) is the most severe problem, less severe drinking problems can also be dangerous. The term "alcoholism" refers to a disease known as alcohol dependence syndrome, the most severe stage of a range of drinking problems.

Symptoms of Alcoholism

Alcoholism is called a progressive disease, meaning that over time the symptoms and effects of drinking alcohol become more intense and severe. The symptoms in the early stages differ from those during later stages as the disease progresses from binge drinking to alcohol abuse to alcohol dependence.

Diagnosing Alcoholism

Because one of the most common symptoms of alcoholism is denial, diagnosing alcoholism can be difficult -- the diagnosis depends on the individual's willingness to answer questions about their drinking honestly. Usually, the friends and family members closest to the drinker see the problem long before it is diagnosed in a medical setting.

Is Alcoholism Inherited?

Alcoholism does tend to run in families, and scientific studies indicate that genetics play a role in a person's risk of developing alcohol problems. But research also shows that a individual's environment and peer influences also affect the risk of becoming alcoholic.

Abuse Signs, Symptoms, and Help for Drug Problems and Substance

Are you struggling with a drug problem that’s spiraled out of control? If so, you may feel isolated, helpless, or ashamed. Or perhaps you’re worried about a friend or family member’s drug use. In either case, you’re not alone. Addiction is a problem that many people face.
The good news is that you or your loved one can get better. There is hope—no matter how bad the substance abuse problem and no matter how powerless you feel. Learning about the nature of addiction—how it develops, what it looks like, and why it has such a powerful hold—will give you a better understanding of the problem and how to deal with it.

How addiction developsThe path to drug addiction starts with experimentation. You or your loved one may have tried drugs out of curiosity, because friends were doing it, or in an effort to erase another problem. At first, the substance seems to solve the problem or make life better, so you use the drug more and more.
But as the addiction progresses, getting and using the drug becomes more and more important and your ability to stop using is compromised. What begins as a voluntary choice turns into a physical and psychological need. The good news is that drug addiction is treatable. With treatment and support, you can counteract the disruptive effects of addiction and regain control of your life.
5 Myths about Drug Addiction and Substance Abuse
MYTH 1: Overcoming addiction is a simply a matter of willpower. You can stop using drugs if you really want to. Prolonged exposure to drugs alters the brain in ways that result in powerful cravings and a compulsion to use. These brain changes make it extremely difficult to quit by sheer force of will.
MYTH 2: Addiction is a disease; there’s nothing you can do about it. Most experts agree that addiction is a brain disease, but that doesn’t mean you’re a helpless victim. The brain changes associated with addiction can be treated and reversed through therapy, medication, exercise, and other treatments.
MYTH 3: Addicts have to hit rock bottom before they can get better. Recovery can begin at any point in the addiction process—and the earlier, the better. The longer drug abuse continues, the stronger the addiction becomes and the harder it is to treat. Don’t wait to intervene until the addict has lost it all.
MYTH 4: You can’t force someone into treatment; they have to want help. Treatment doesn’t have to be voluntary to be successful. People who are pressured into treatment by their family, employer, or the legal system are just as likely to benefit as those who choose to enter treatment on their own. As they sober up and their thinking clears, many formerly resistant addicts decide they want to change.
MYTH 5: Treatment didn’t work before, so there’s no point trying again; some cases are hopeless. Recovery from drug addiction is a long process that often involves setbacks. Relapse doesn’t mean that treatment has failed or that you’re a lost cause. Rather, it’s a signal to get back on track, either by going back to treatment or adjusting the treatment approach.

The far-reaching effects of drug abuse and drug addictionWhile each drug of abuse produces different physical effects, all abused substances share one thing in common. They hijack the brain’s normal “reward” pathways and alter the areas of the brain responsible for self-control, judgment, emotional regulation, motivation, memory, and learning.
Whether you’re addicted to nicotine, alcohol, heroin, Xanax, speed, or Vicodin, the effect on the brain is the same: an uncontrollable craving to use that is more important than anything else, including family, friends, career, and even your own health and happiness.
Using drugs as an escape: A short-term fix with long-term consequences
Many people use drugs in order to escape physical and emotional discomfort. Maybe you started drinking to numb feelings of depression, smoking pot to deal with stress at home or school, relying on cocaine to boost your energy and confidence, using sleeping pills to cope with panic attacks, or taking prescription painkillers to relieve chronic back pain.
But while drugs might make you feel better in the short-term, attempts to self-medicate ultimately backfire. Instead of treating the underlying problem, drug use simply masks the symptoms. Take the drug away and the problem is still there, whether it be low self-esteem, anxiety, loneliness, or an unhappy family life. Furthermore, prolonged drug use eventually brings its own host of problems, including major disruptions to normal, daily functioning. Unfortunately, the psychological, physical, and social consequences of drug abuse and addiction become worse than the original problem you were trying to cope with or avoid.

Signs and symptoms of drug abuse and drug addiction
Although different drugs have different physical effects, the symptoms of addiction are the same no matter the substance. The more drugs begin to affect and control your life, the more likely it is that you’ve crossed the line from drug use to abuse and drug addiction. Unfortunately, when you’re in the middle of it, you may be in denial about the magnitude of the problem or the negative impact it's had on your life. See if you recognize yourself in the following signs and symptoms of substance abuse and addiction. If so, consider talking to someone about your drug use. You’re on a dangerous road, and the sooner you get help, the better.
Common signs and symptoms of drug abuse
You’re neglecting your responsibilities at school, work, or home (e.g. flunking classes, skipping work, neglecting your children) because of your drug use.
You’re using drugs under dangerous conditions or taking risks while high, such as driving while on drugs, using dirty needles, or having unprotected sex.
Your drug use is getting you into legal trouble, such as arrests for disorderly conduct, driving under the influence, or stealing to support a drug habit.
Your drug use is causing problems in your relationships, such as fights with your partner or family members, an unhappy boss, or the loss of old friends.
Common signs and symptoms of drug addiction
You’ve built up a drug tolerance. You need to use more of the drug to experience the same effects you used to with smaller amounts.
You take drugs to avoid or relieve withdrawal symptoms. If you go too long without drugs, you experience symptoms such as nausea, restlessness, insomnia, depression, sweating, shaking, and anxiety.
You’ve lost control over your drug use. You often do drugs or use more than you planned, even though you told yourself you wouldn’t. You may want to stop using, but you feel powerless.
Your life revolves around drug use. You spend a lot of time using and thinking about drugs, figuring out how to get them, and recovering from the drug’s effects.
You’ve abandoned activities you used to enjoy, such as hobbies, sports, and socializing, because of your drug use.
You continue to use drugs, despite knowing it’s hurting you. It’s causing major problems in your life—blackouts, infections, mood swings, depression, paranoia—but you use anyway.
What drugs are most commonly abused and what are the signs and symptoms?
Almost all drugs have the potential for addiction and abuse, from caffeine to prescription medication. However, the majority of non-alcohol related addictions are due to a short list of drugs including sleeping pills, painkillers, cocaine, marijuana, methamphetamine, and heroin.
Click here for a PDF factsheet on the symptoms and effects of commonly abused drugs.
Warning signs that a friend or family member is abusing drugs
Drug abusers often try to conceal their symptoms and downplay their problem. If you’re worried that a friend or family member might be abusing drugs, look for the following warning signs:
Physical warning signs of drug abuse
Bloodshot eyes or pupils that are larger or smaller than usual.
Changes in appetite or sleep patterns. Sudden weight loss or weight gain.
Deterioration of physical appearance and personal grooming habits.
Unusual smells on breath, body, or clothing.
Tremors, slurred speech, or impaired coordination.
Behavioral signs of drug abuse
Drop in attendance and performance at work or school.
Unexplained need for money or financial problems. May borrow or steal to get it.
Engaging in secretive or suspicious behaviors.
Sudden change in friends, favorite hangouts, and hobbies.
Frequently getting into trouble (fights, accidents, illegal activities).
Psychological warning signs of drug abuse
Unexplained change in personality or attitude.
Sudden mood swings, irritability, or angry outbursts.
Periods of unusual hyperactivity, agitation, or giddiness.
Lack of motivation; appears lethargic or “spaced out.”
Appears fearful, anxious, or paranoid, with no reason.
Warning Signs of Teen Drug Use
There are many warning signs of drug use and abuse in teenagers. The challenge for parents is to distinguish between the normal, sometimes volatile, ups and downs of the teen years and the red flags of substance abuse.
Being secretive about friends, possessions, and activities.
New interest in clothing, music, and other items that highlight drug use.
Demanding more privacy; locking doors; avoiding eye contact; sneaking around.
Skipping class; declining grades; suddenly getting into trouble at school.
Missing money, valuables, or prescriptions.
Acting uncharacteristically isolated, withdrawn, or depressed.
Using incense, perfume, or air freshener to hide the smell of smoke or drugs.
Using eyedrops to mask bloodshot eyes or dilated pupils.

Drug addiction and denial
One of the most dangerous effects of drug abuse and addiction is denial. The urge to use is so strong that the mind finds many ways to rationalize the addiction. You may drastically underestimate the quantity of drugs you’re taking, how much it impacts your life, and the level of control you have over your drug use.
Denial is an unconscious defense mechanism. Minimizing and rationalizing the addiction is less scary than admitting that your drug use is dangerously out of control. But the cost of denial can be extremely high—including the loss of important relationships, your job, financial security, and your physical and mental health.

Do you have a substance abuse problem?
Do you feel like you can’t stop, even if you wanted to?
Do you ever feel bad or guilty about your drug use?
Do you need to use drugs to relax or feel better?
Do your friends or family members complain or worry about your drug use?
Do you hide or lie about your drug use?
Have you ever done anything illegal in order to obtain drugs?
Do you spend money on drugs that you really can’t afford?
Do you ever use more than one recreational drug at a time?
If you answered “yes” to one or more of the questions, you may have a drug problem.

Getting help for drug abuse and drug addiction

If you’re ready to admit you have a drug problem, congratulations! Recognizing that you have a problem is the first step on the road to recovery, one that takes tremendous courage and strength.
Facing your addiction without minimizing the problem or making excuses can feel frightening and overwhelming, but recovery is within reach. If you’re ready to make a change and willing to seek help, you can overcome your addiction and build a satisfying, drug-free life for yourself.
Support is essential to addiction recovery
Don’t try to go it alone; it’s all too easy to get discouraged and rationalize “just one more” hit or pill. Whether you choose to go to rehab, rely on self-help programs, get therapy, or take a self-directed treatment approach, support is essential. Recovering from drug addiction is much easier when you have people you can lean on for encouragement, comfort, and guidance

LUCIFERASE GENE AND ITS APPLICATIONS

Background
Bioluminescence is a form of light produced by a chemical reaction in living
organisms. The function of bioluminescence may vary from one organism to the other,
such as for defense against predators, for predation or for communication with their
mates . It is exhibited by a diverse group of organisms
although their number is very less compared to the total number of known species. It has
been estimated that luminous organisms may have come from about 30 different
evolutionarily distinct origins .
The enzyme luciferase and its substrate luciferin are discovered to be the agents
responsible for Bioluminescence. The biochemical and physiological mechanisms
responsible for the bioluminescence are different among the luminous organisms
. Hastings and Morin in 1991 have reported that the genes and
proteins responsible for bioluminescence are not evolutionarily conserved. These genes
and proteins are evolved independently and are not similar to each other.
Shimomura et al., first discovered luciferase in 1962. They extracted the light
emitting protein from jellyfish “Aequorea aequorea” and found the compound to be
emitting blue color instead of expected green color. This protein was later named as
“aequorin”. The expected green color was emitted by another protein, which absorbed the
blue color emitted by aequorin and re-emitted the energy at a higher wavelength. This
protein was later named “green fluorescence protein” (GFP).
There have been many studies on luciferase enzyme systems in different groups
of luminescent organisms. There are also many applications of this enzyme and the
associated GFP in molecular biology fields. In the following review the different aspects
of the enzyme luciferase and its applications will be addressed.
Chemistry
The chemistry behind bioluminescence has two major components. One is the
substrate which is generically called luciferin and the other is the enzyme that catalyses
the substrate to emit a photon called as luciferase.

The basic reaction involves luciferase catalyzing the oxidation of luciferin
resulting in light and an inactive "oxyluciferin" In most cases, fresh luciferin must be
brought into the system, either through the diet or by internal synthesis.
Luciferin + O2 Oxyluciferin + light
Luciferase
Sometimes the luciferin and luciferase (as well as a co-factor such as oxygen) are
bound together in a single unit called a "photoprotein". This molecule can be triggered to
produce light when a particular type of ion is added to the system (frequently
calcium).

Types of Luciferin
Luminous organisms are intensively studied for their difference in the luminous
systems. The luminous systems differ in the structures of the luciferins and luciferases,
although they have some properties in common. All known luciferases are oxygenases
and they oxidize the luciferin using molecular oxygen to intermediate enzyme bound
peroxide, which dissociates to produce an intermediate or product in its excited state to
emit photon. Luminous systems of luminous organisms are classified into four types
based on the different type of luciferin they possess. They are 1) Coelenterate luciferin
(Coelenterazine), 2) Bacterial luciferin, 3) Dinoflagellate luciferin, and 4) Firefly
luciferin.
Coelenterazine is the most popular of the marine luciferins, found in a variety of
phyla. This luciferin is biochemically similar to other cnidarian luminescent systems. It
possesses an imidazopyrazine skeleton and is widely distributed among the cnidarian
group.
Coelenterazine
Bacterial luciferin is a reduced riboflavin phosphate, which is oxidized in
association with a long-chain aldehyde, oxygen, and a luciferase. Luminous bacteria are
observed ubiquitously in seawater samples. All bacterial luciferases are heterodimeric
containing two polypeptides alpha and beta. Fisher et al. in 1995 reported the X-ray
structure of luciferases.
Bacterial Luciferin

Dinoflagellate luciferin is a novel tetrapyrrole related to chlorophyll. In the genus
Gonyaulax, at pH 8 the molecule is "protected" from the luciferase by a "luciferinbinding
protein", but when the pH lowers to around 6, the free luciferin reacts and light is
produced. A modified form of this luciferin is also found in herbivorous euphausiid
shrimp, perhaps indicating a dietary link for the acquisition of luciferin.

Firefly luciferin is a unique benzothiazolethat requires ATP to form luciferyl
adenylate intermediate. The adenylate then reacts with with oxygen to form a cyclic
luciferyl perooxy speces, which breaks down to yield CO2 and an excited state of the
carbonyl product. Wood in 1995 reported that the long chain acyl-CoA synthase is
homologous with the firefly luciferase indicating the evolutionary origin of the gene

Luciferase Gene organisation
Bacterial luciferase gene
Luciferase genes are cloned and sequenced from luminescent bacteria of Vibrio,
Xenorhabdus and Photobacterium genera (Szittner, 1990). Two genes code for the two á
and â subunits of bacterial luciferase , named luxA and luxB respectively. The
luciferase genes occur as two major operons, also containing other proteins associated
with the luciferase activity that is involved in the synthesis of myristic aldehyde
(Meighen, 1991) . The operon also contains genes that control the development
and expression of luminescence by a mechanism of “autoinduction” (Nealson et al.,
1970). The luciferase operon contains about 10 genes and they are transcribed only by the
presence of an autoinducer which is a homoserine lactone produced by the gene present
6
in the operon itself named luxI gene. The ecological importance of this mechanism was
discovered in 1994 by Fuqua et al., who dubbed this phenomenon to be Quarum sensing.
It is also discovered that the autoinducer is responsible for the cell-cell signaling in
luminous bacteria.
Plate 2 Bacterial luciferase

Dinoflagellate luciferase gene
The Gonyaulax polyedra luciferin binding protein (LBP) gene and the luciferase
gene are the first dinoflagellate luciferase gene cloned and sequenced by Lee et al.,
(1993) and Bae and Hastings (1994) respectively. They sequenced the cDNA of the LBP
gene and found that the open reading frame of the gene is 2004 nucleotides in length and
has no introns. It is also found that it has no polyadenylation sites. The open reading
frame codes for about 688 aminoacids (~ 75kDa). The interesting circadian phenomenon
is seen in the dinoflagellate LBP gene, which causes the gene to be transcribed only once
a day and the protein destroyed every day (Hastings et al., 1993).
The luciferase gene was found to be 4.1kb and the luciferase enzyme is 130kDa.
This gene also did not have any introns. Cloning of the cDNA of the gene in Escherichia
coli showed the expression of the active luciferase protein (Bae and Hastings, 1994). It
was found that the LBP is responsible for the pH dependent binding of luciferin and the
luciferase is responsible for the oxidation of the luciferin to produce light. Li et al.,
(1997) reported a unique case of a 137kDa protein gene responsible of catalyzing the
light-emitting oxidation of the dinoflagellate luciferin. They also found that the gene had
three tandem repeat sequences of (~377, 377 and 379 bp) with no intervals, capable of
producing individually active luciferase.

Okamoto et al., in 2001 reported three new classes of the luciferase gene in new
members of dinoflagellate, Pyrocystis lunula and Lingulodinium polyedrum. The three
genes are named lcfA, lcfB and lcfC, which are unique and not related to the luciferase
genes of other dinoflagellates. The lcfA and lcfB genes are connected by a 2.2kb
intergenic region. In P.lunula, the lcfC gene has an intron (Figure 5). They also found the
presence of an intron in lcfC of P. lunula luciferase genes; different sizes of untranslated
regions in P. lunula and L. polyedrum luciferase genes, sharing no significant sequence
similarities; and notable differences in the frequency of synonymous substitutions in the
center of the repeat luciferase domains between the two species

Firefly luciferase gene
The Firefly luciferase gene was first cloned and sequenced by Wet et al., in 1985
from Photuris pennsylvanica, belonging to the Photurinae subfamily of the Lampyridae.
The gene sequence was about 1.8kb in length, coding for 545 aminoacid . The 5'
noncoding region was 61 bp and the 3' noncoding region is 135 bp in length. The
poly(A)tail was 24-nucleotide in length (Figure 6). Wood in 1995 studied the homology
of the luciferase enzyme aminoacid sequences with other known enzyme sequences. He
showed the enzyme’s similarity with adenylate kinase, the putative AMP-binding
domain, luciferin 4-monooxygenase, 4-coumarate CoA ligase, long-chain fatty acid CoA
ligase, 2-acylglycerophosphoethanolamine acyltransferase, the microbody-directing
sequence, peptide-synthesizing complexes, and acyladenylate-synthesizing enzymes.
Choi et al., in 2003 studied the luciferase gene organization in the Hotaria-group of
fireflies. They also studied the phylogenetic diversity of the luciferase gene in fireflies of
the Hotaria group.
Plate

Applications of Luciferase
Initial applications of luciferase were dependent on the type substrate that were
used for emitting light. The apoaequorin, which functions like the luciferase in
coelenterate system, was used to monitor intracellular calcium levels (Tanahashi et al.,
1990). Saran et al., (1994) used cnidarian luciferases to monitor intracellular calcium
changes in response to cyclic adenosinemonophosphate (cAMP) stimulation. Johnson et
al., (1995) used coelenterate luciferased to investigate the circadian changes in free
cytosolic calcium level in the chloroplasts of tobacco and Arabidopsis. Similarly bacterial
lux genes have been used as reporters for visualizing gene expression in Streptomyces
(Schauer et al., 1988) and to study the circadian transcriptional regulation in
cyanobacteria (Kondo et al., 1994). The firefly luciferase was widely used to measure
ATP (Hastings et al., 1997). Recently a combined use of two different luminous systems,
10
such as firefly and Renilla luciferase enzymes are used to assay two different luciferins
has been reported by Sherf et al., 1997.
Luciferases are nowadays successfully used as reporter genes for gene expression
studies in living cells. Their application is growing in all aspects of the biological field
such as Animal research, environmental and plant research.
Luciferase in Animal Research
Molecular imaging of live cells
Luciferase is widely used in molecular imaging of cells in vivo. Luciferases are
used to track the in vivo behaviour of any tissue. Bhaumik and Gambhir (2002) validated
that the bioluminescence from Renilla luciferase (rluc) and the firefly luciferase enzyme
both can be imaged using different substrates for each and their kinetics can be studied in
vivo in the tissue. They carried out the experiment in mice by transfecting the mice with
C6 cells containing Firefly luciferase (fluc) and (rluc) (Plate 4).
Plate 4 Kinetics of light production from mice carrying s.c. C6-Fluc and C6-Rluc cells after simultaneous tail-vein injection of both Dluciferin
and coelenterazine. A mouse was injected s.c. with C6-Fluc (A), C6-Rluc (B), and C6 control cells (C) on right forearm, left
forearm, and right thigh regions, respectively. Simultaneous injection of both coelenterazine and D-luciferin mixture via tail-vein
shows bioluminescence from both the sites simultaneously but with distinct kinetics. A series of image at 2-min intervals is shown
from the same mouse. Each image represents a scan time of 1 min. The signal from C6-Rluc cells (B) peaks early and is near
extinguished within 10 min. Bioluminescence from C6-Fluc cells (A) shows a relatively strong signal beyond 10 min. The region of
control cells does not show any significant bioluminescence. R and L represent the right and left side of the mouse resting in supine
position.
Applications in Gene therapies
Molecular imaging of reporter therapeutic genes could play a critical role in gene
therapy to achieve controlled and effective delivery of genes to target cells avoiding
ectopic expression. Luciferases play a major role in reporting the level, the location and
the duration of the gene expression. A study by Weng et al., (2000) used somatic cell
gene transfer of the fluc linked heme oxygenase-1 (HO-1) transgene to the lung alveoli of
neonatal mice through transpulmonary injection. They monitored the gene expression in
11
real time and were able to detect targeted expression of the gene in the cells of the
alveolar epithelium rather than other cells of the lung

Application in transgenic animal research
A study carried out by Voojis et al., (2002) involved the use of a conditional
mouse model for retinoblastoma suppressor gene-dependent pituitary cancer development
with co-expression of fluc genes enabling long term bioluminescence imaging,
quantification of tumour and assessment of chemotherapeutic response(Plate 6) .
Plate 6 Pseudocolor images of longitudinal tumor growth in a POMCcre-POMCluc;RbF19/F19 animal.
Quantification of theemitted photons shows an approximately exponential tumor growth between 12 and 16 weeks.
Another study carried out by Carlsen et al., (2002) developed transgenic mice that
express the fluc gene under the control of the nuclear factor 6B (NF-6B) promoter. This
enabled then for real-time imaging of the NF-6B promoter activity and its modulation in
living mice (Plate 7

Luciferases in Environmental research
Applications as biosensors
Luciferase reporter systems are developed into luminescent bacterial biosensors to
detect a wide range of pollutants and also to assess the bioavailability in the
environmental samples. Hollis et al., in 1999 transformed a Saccharomyces cerevisiae
with firefly luciferase gene from Photinus pyralis to be used as a biosensor. They studied
the cell health upon exposure to toxins directly correlating with the endogenous supply of
energy by ATP. A bioluminescent plasmid reporter that shows a sensitive response
towards 2-4-Dichlorophenoxyacetic acid and 2, 4-Dichlorophenol in soil was constructed
and introduced into the chromosome of Ralstonia eutropha. The transformed bacterium is
capable of detecting the concentrations of 1.2mM 2,4-D and 1.1X102 μM of 2,4-
dichlorophenol (Hay et al., 2000). Pellinen et al., (2002) developed Escherichia coli K-12
strain for specific detection of the tetracycline family of antimicrobial agents. It was
optimized to work with fish samples. The biosensing strain contains a plasmid
incorporating the bacterial luciferase operon of Photorhabdus luminescens under the
control of the tetracycline responsive element from transposon Tn10. The lowest levels of
detection of tetracycline and oxytetracycline from spiked fish tissue were 20 and 50
μg/kg, respectively, in a 2-h assay.

Transgenic Animals

A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. In addition to a structural gene, the DNA usually includes other sequences to enable it
to be incorporated into the DNA of the host and
to be expressed correctly by the cells of the host.
Transgenic sheep and goats have been produced that express foreign proteins in their milk.
Transgenic chickens are now able to synthesize human proteins in the "white" of the eggs. These animals should eventually prove to be valuable sources of proteins for human therapy.
In July 2000, researchers from the team that produced Dolly reported success in producing transgenic lambs in which the transgene had been inserted at a specific site in the genome and functioned well. [More]
Transgenic mice have provided the tools for exploring many biological questions.
An example:Normal mice cannot be infected with polio virus. They lack the cell-surface molecule that, in humans, serves as the receptor for the virus. So normal mice cannot serve as an inexpensive, easily-manipulated model for studying the disease. However, transgenic mice expressing the human gene for the polio virus receptor
can be infected by polio virus and even
develop paralysis and other pathological changes characteristic of the disease in humans.
Two methods of producing transgenic mice are widely used:
transforming embryonic stem cells (ES cells) growing in tissue culture with the desired DNA;
injecting the desired gene into the pronucleus of a fertilized mouse egg.
The Embryonic Stem Cell Method (Method "1")Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts. They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes.

. Make your DNAUsing recombinant DNA methods, build molecules of DNA containing
the structural gene you desire (e.g., the insulin gene)
vector DNA to enable the molecules to be inserted into host DNA molecules
promoter and enhancer sequences to enable the gene to be expressed by host cells
2. Transform ES cells in cultureExpose the cultured cells to the DNA so that some will incorporate it.
3. Select for successfully transformed cells. [Method]
4. Inject these cells into the inner cell mass (ICM) of mouse blastocysts.
5. Embryo transfer
Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male). The stimulus of mating elicits the hormonal changes needed to make her uterus receptive.
Transfer the embryos into her uterus.
Hope that they implant successfully and develop into healthy pups (no more than one-third will).
6. Test her offspring
Remove a small piece of tissue from the tail and examine its DNA for the desired gene. No more than 10–20% will have it, and they will be heterozygous for the gene.
7. Establish a transgenic strain
Mate two heterozygous mice and screen their offspring for the 1:4 that will be homozygous for the transgene.
Mating these will found the transgenic strain.

The Pronucleus Method (Method "2")
1. Prepare your DNA as in Method 1
2. Transform fertilized eggs
Harvest freshly fertilized eggs before the sperm head has become a pronucleus.
Inject the male pronucleus with your DNA.
When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide by mitosis to form a 2-cell embryo.
3. Implant the embryos in a pseudopregnant foster mother and proceed as in Method 1.
An Example This image (courtesy of R. L. Brinster and R. E. Hammer) shows a transgenic mouse (right) with a normal littermate (left). The giant mouse developed from a fertilized egg transformed with a recombinant DNA molecule containing:
the structural gene for human growth hormone
a strong mouse gene promoterThe levels of growth hormone in the serum of some of the transgenic mice were several hundred times higher than in control mice.
Random vs. Targeted Gene InsertionThe early vectors used for gene insertion could, and did, place the gene (from one to 200 copies of it) anywhere in the genome. However, if you know some of the DNA sequence flanking a particular gene, it is possible to design vectors that replace that gene. The replacement gene can be one that
restores function in a mutant animal or
knocks out the function of a particular locus.In either case, targeted gene insertion requires
the desired gene
neor, a gene that encodes an enzyme that inactivates the antibiotic neomycin and its relatives, like the drug G418, which is lethal to mammalian cells;
tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside analog gancyclovir. DNA polymerase fails to discriminate against the resulting nucleotide and inserts this nonfunctional nucleotide into freshly-replicating DNA. So ganciclovir kills cells that contain the tk gene.
Step 1Treat culture of ES cells with preparation of vector DNA.
Results:
Most cells fail to take up the vector; these cells will be killed if exposed to G418.
In a few cells: the vector is inserted randomly in the genome. In random insertion, the entire vector, including the tk gene, is inserted into host DNA. These cells are resistant to G418 but killed by gancyclovir.
In still fewer cells: homologous recombination occurs. Stretches of DNA sequence in the vector find the homologous sequences in the host genome and the region between these homologous sequences replaces the equivalent region in the host DNA.
Step 2Culture the mixture of cells in medium containing both G418 and ganciclovir.
The cells (the majority) that failed to take up the vector are killed by G418.
The cells in which the vector was inserted randomly are killed by gancyclovir (because they contain the tk gene).
This leaves a population of cells transformed by homologous recombination (enriched several thousand fold).
Step 3Inject these into the inner cell mass of mouse blastocysts.
Knockout Mice: What do they teach us?
If the replacement gene (A* in the diagram) is nonfunctional (a "null" allele), mating of the heterozygous transgenic mice will produce a strain of "knockout mice" homozygous for the nonfunctional gene (both copies of the gene at that locus have been "knocked out"). Knockout mice are valuable tools for discovering the function(s) of genes for which mutant strains were not previously available. Two generalizations have emerged from examining knockout mice:
Knockout mice are often surprisingly unaffected by their deficiency. Many genes turn out not to be indispensable. The mouse genome appears to have sufficient redundancy to compensate for a single missing pair of alleles.
Most genes are pleiotropic. They are expressed in different tissues in different ways and at different times in development.
Tissue-Specific Knockout Mice

While "housekeeping" genes are expressed in all types of cells at all stages of development, other genes are normally expressed in only certain types of cells when turned on by the appropriate signals (e.g. the arrival of a hormone).
Link to a discussion of cell-specific gene expression.
To study such genes, one might expect that the methods described above would work. However, it turns out that genes that are only expressed in certain adult tissues may nonetheless be vital during embryonic development. In such cases, the animals do not survive long enough for their knockout gene to be studied. Fortunately, there are now techniques with which transgenic mice can be made where a particular gene gets knocked out in only one type of cell.
The Cre/loxP System
One of the bacteriophages that infects E. coli, called P1, produces an enzyme — designated Cre — that cuts its DNA into lengths suitable for packaging into fresh virus particles. Cre cuts the viral DNA wherever it encounters a pair of sequences designated loxP. All the DNA between the two loxP sites is removed and the remaining DNA ligated together again (so the enzyme is a recombinase). Using "Method 1" (above), mice can be made transgenic for
the gene encoding Cre attached to a promoter that will be activated only when it is bound by the same transcription factors that turn on the other genes required for the unique function(s) of that type of cell;
a "target" gene, the one whose function is to be studied, flanked by loxP sequences. In the adult animal,
those cells that
receive signals (e.g., the arrival of a hormone or cytokine)
to turn on production of the transcription factors needed
to activate the promoters of the genes whose products are needed by that particular kind of cellwill also turn on transcription of the Cre gene. Its protein will then remove the "target" gene under study.
All other cells will lack the transcription factors needed to bind to the Cre promoter (and/or any enhancers) so the target gene remains intact.
The result: a mouse with a particular gene knocked out in only certain cells.
Knock-in Mice
The Cre/loxP system can also be used to
remove DNA sequences that block gene transcription. The "target" gene can then be turned on in certain cells or at certain times as the experimenter wishes.
replace one of the mouse's own genes with a new gene that the investigator wishes to study.
Such transgenic mice are called "knock-in" mice.
Transgenic Sheep and Goats
Until recently, the transgenes introduced into sheep inserted randomly in the genome and often worked poorly. However, in July 2000, success at inserting a transgene into a specific gene locus was reported. The gene was the human gene for alpha1-antitrypsin, and two of the animals expressed large quantities of the human protein in their milk.
This is how it was done.
Sheep fibroblasts (connective tissue cells) growing in tissue culture were treated with a vector that contained these segments of DNA:
2 regions homologous to the sheep COL1A1 gene. This gene encodes Type 1 collagen. (Its absence in humans causes the inherited disease osteogenesis imperfecta.)
This locus was chosen because fibroblasts secrete large amounts of collagen and thus one would expect the gene to be easily accessible in the chromatin.
A neomycin-resistance gene to aid in isolating those cells that successfully incorporated the vector. [Link to technique]
The human gene encoding alpha1-antitrypsin.
Some people inherit two non- or poorly-functioning genes for this protein. Its resulting low level or absence produces the disease Alpha1-Antitrypsin Deficiency (A1AD or Alpha1). The main symptoms are damage to the lungs (and sometimes to the liver).
Promoter sites from the beta-lactoglobulin gene. These promote hormone-driven gene expression in milk-producing cells.
Binding sites for ribosomes for efficient translation of the mRNAs. Successfully-transformed cells were then
fused with enucleated sheep eggs [Link to description of the method] and
implanted in the uterus of a ewe (female sheep).
Several embryos survived until their birth, and two young lambs have now lived over a year.
When treated with hormones, these two lambs secreted milk containing large amounts of alpha1-antitrypsin (650 µg/ml; 50 times higher than previous results using random insertion of the transgene).
On June 18, 2003, the company doing this work abandoned it because of the great expense of building a facility for purifying the protein from sheep's milk. Purification is important because even when 99.9% pure, human patients can develop antibodies against the tiny amounts of sheep proteins that remain. However, another company, GTC Biotherapeutics, has persevered and in June of 2006 won preliminary approval to market a human protein, antithrombin, in Europe. Their protein — the first made in a transgenic animal to receive regulatory approval for human therapy — was secreted in the milk of transgenic goats.
Transgenic ChickensChickens
grow faster than sheep and goats and large numbers can be grown in close quarters;
synthesize several grams of protein in the "white" of their eggs. Two methods have succeeded in producing chickens carrying and expressing foreign genes.
Infecting embryos with a viral vector carrying
the human gene for a therapeutic protein
promoter sequences that will respond to the signals for making proteins (e.g. lysozyme) in egg white.
Transforming rooster sperm with a human gene and the appropriate promoters and checking for any transgenic offspring.
Preliminary results from both methods indicate that it may be possible for chickens to produce as much as 0.1 g of human protein in each egg that they lay.
Not only should this cost less than producing therapeutic proteins in culture vessels, but chickens will probably add the correct sugars to glycosylated proteins — something that E. coli cannot do.
Transgenic Pigs
Transgenic pigs have also been produced by fertilizing normal eggs with sperm cells that have incorporated foreign DNA. This procedure, called sperm-mediated gene transfer (SMGT) may someday be able to produce transgenic pigs that can serve as a source of transplanted organs for humans. [More]
Transgenic Primates
In the 28 May 2009 issue of Nature, Japanese scientists report success in creating transgenic marmosets. Marmosets are primates and thus our closest relatives (so far) to be genetically engineered. In some cases, the transgene (for green fluorescent protein) was incorporated into the germline and passed on to the animal's offspring. The hope is that these transgenic animals will provide the best model yet for studying human disease and possible therapies

Transgenic plants

Transgenic plants are plants possessing a single or multiple genes, transferred from a different species. Though DNA from another species can be integrated into a plants' genome via natural processes, the term "transgenic plants" refers to plants created in a laboratory using recombinant DNA technology.

The aim of creating transgenic plants is to design plants with specific characteristics through artificial insertion of genes from other species (or taxonomically up to different kingdoms).

Varieties containing genes of two distinct plant species are frequently created by classical breeders who deliberately force hybridization between distinct plant species when carrying out interspecific or intergeneric wide crosses with the intention of developing disease resistant crop varieties.

Classical plant breeders use a number of in vitro techniques such as protoplast fusion, embryo rescue or mutagenesis to generate diversity and produce plants that would not ordinarily exist in nature (see also Plant breeding, Heterosis, New Rice for Africa).

Such traditional techniques (used from ca 1930) have never been controversial[opinion], or been given wide publicity except among professional biologists, and have allowed crop breeders to develop varieties of basic food crop. Hope is one such wheat variety bred by E. S. McFadden with a gene from a wild grass to have rust resistance. Hope saved American wheat growers from devastating stem rust outbreaks in the 1930s[citation needed].

Methods used in traditional breeding that generate plants with DNA from two species by non-recombinant methods are widely familiar to professional plant scientists, and serve important roles in securing a sustainable future for agriculture by protecting crops from pests and helping land and water to be used more efficiently.[citation needed] (see also Food security, International Fund for Agricultural Development, International development)

Natural gene flow between species

Natural flow of genes between bacterial species, often called horizontal gene transfer or lateral gene transfer, can occur because of gene transfer mediated by natural processes.
This natural gene movement between bacteria has been widely detected during genetic investigation of various natural mobile genetic elements, such as transposons, and retrotransposons that naturally translocate to new sites in a genome, and often move to new species over an evolutionary time scale.
There are many types of natural mobile DNAs, and they have been detected abundantly in food crops such as rice .
These various mobile genes play a major role in dynamic changes to chromosomes during evolution and have often been given whimsical names, such as Mariner, Hobo, Trans-Siberian Express (Transib), Osmar, Helitron, Sleeping Princess, MITE and MULE, to emphasize their mobile and transient behavior.
Genetically mobile DNA constitutes a major fraction of the DNA of many plants, and the natural dynamic changes to crop plant chromosomes caused by this natural transgenic DNA mimics many of the features of plant genetic engineering currently pursued in the laboratory, such as using transposons as a genetic tool, and molecular cloning. See also transposon, retrotransposon, integron, provirus, endogenous retrovirus, heterosis, Gene duplication and exon shuffling by helitron-like transposons generate intraspecies diversity in maize.
There is new scientific literature about natural transgenic events in plants, through movement of natural mobile DNAs called MULEs between rice and Setaria millet.
It is becoming clear that natural rearrangements of DNA and horizontal gene transfer play a pervasive role in natural evolution. Importantly many, if not most, flowering plants evolved by transgenesis[citation needed] - that is, the creation of natural interspecies hybrids in which chromosome sets from different plant species were added together. There is also the long and rich history of interspecies cross-breeding with traditional methods.

Deliberate creation of transgenic plants
Production of transgenic plants in wide-crosses by plant breeders has been a vital aspect of conventional plant breeding for about a century[citation needed]. Without it, security of our food supply against losses caused by crop pests such as rusts and mildews would be severely compromised[citation needed]. The first historically recorded interspecies transgenic cereal hybrid was actually between wheat and rye .
In the 20th century, the introduction of alien germplasm into common foods was repeatedly achieved by traditional crop breeders by artificially overcoming fertility barriers. Novel genetic rearrangements of plant chromosomes, such as insertion of large blocks of rye (Secale) genes into wheat chromosomes ('translocations'), has also been exploited widely for many decades [5].
By the late 1930s with the introduction of colchicine, perennial grasses were being hybridized with wheat with the aim of transferring disease resistance and perenniality into annual crops, and large-scale practical use of hybrids was well established, leading on to development of Triticosecale and other new transgenic cereal crops. In 1985 Plant Genetic Systems (Ghent, Belgium), founded by Marc Van Montagu and Jeff Schell, was the first company to develop genetically engineered (tobacco) plants with insect tolerance by expressing genes encoding for insecticidal proteins from Bacillus thuringiensis (Bt).

Transgenic resistance traits in bread wheat varieties

The lists in this article may contain items that are not notable, encyclopedic, or helpful. Please help out by removing such elements and incorporating appropriate items into the main body of the article. (January 2010)
Important transgenic pathogen and parasite resistance traits in current bread wheat varieties (gene, eg "Lr9" followed by the source species) are:
Disease resistance
Leaf rust
Lr9 (from Aegilops umbellulata)
Lr18 Triticum timopheevi
Lr19 Thinopyrum
Lr23 T. turgidum
Lr24 Ag. elongatum
Lr25 Secale cereale
Lr29 Ag. elongatum
Lr32 T. tauschii
Stem rust
Sr2 T. turgidum ("Hope" ) McFadden, E. S. (1930) J. Am. Soc. Agron. 22, 1020-1031 .
Sr22 Triticum monococcum
Sr36 Triticum timopheevii
Stripe rust
Yr15 Triticum dicoccoides
Powdery mildew
Pm12 Aegilops speltoides
Pm21 Haynaldia villosa
Pm25 T. monococcum
Wheat streak mosaic virus
Wsm1 Ag. elongatum
Pest resistance
Hessian fly
H21 S. cereale H23,
H24 T. tauschii
H27 Aegilops ventricosa
Cereal cyst nematode
Cre3 (Ccn-D1) T. tauschii
Lepidoptera
Bt Bacillus thuringiensis

Genetically engineered plants

Plums that have been genetically engineered to be resistant to the plum pox virus
The intentional creation of transgenic plants by laboratory based recombinant DNA methods is more recent (from the mid-70s on[citation needed]) and has been a controversial development in the field of biotechnology opposed vigorously by many NGOs, and several governments, particularly in Europe.
These transgenic recombinant plants are transforming agriculture in those regions that have allowed farmers to adopt them, and the area sown to these crops has continued to grow globally in every years since their first introduction in 1996.
As of 2006 there were around 250 million acres of genetically engineered crops being grown commercially in 22 countries[citation needed]. The U.S. has adopted the technology most widely whereas Europe has almost no genetically engineered crops. The EU had a formal ban on GM crops, until it was overturned in 2006; in a controversial move GM crops are now regulated by the EU.Transgenic recombinant plants are generated in a laboratory by adding one or more genes to a plant's genome,and the techniques frequently called transformation. Transformation is usually achieved using gold particle bombardment or through the process of Horizontal gene transfer using a soil bacterium, Agrobacterium tumefaciens, carrying an engineered plasmid vector, or carrier of selected extra genes.
Transgenic recombinant plants are identified as a class of genetically modified organism(GMO); usually only transgenic plants created by direct DNA manipulation are given much attention in public discussions[opinion].
Transgenic plants have been deliberately developed for a variety of reasons: longer shelf life, disease resistance, herbicide resistance, pest resistance, non-biological stress resistances, (e.g. drought or nitrogen starvation), and nutritional improvement (see Golden rice) and frost tolerance (see Fish tomato).
The first modern recombinant crop approved for sale in the U.S., in 1994, was the FlavrSavr tomato, which had a longer shelf life. The first conventional transgenic cereal created by scientific breeders was actually a hybrid between wheat and rye (triticale) in 1876 (Wilson, 1876). The first transgenic cereal may have been wheat, which itself is a natural transgenic plant derived from at least three parental species.
Genetically modified organisms came before commercially viable crops as the FlavrSavr tomato, only strictly grown indoors (in laboratories). However, after the introduction of the Flavr Savr tomato, certain GMO-crops (e.g. GMO-soy, GMO-corn, etc.) in the US were being grown outdoors on large scales.
Commercial factors, including high regulatory and research costs, have so far restricted modern transgenic crop varieties to major traded commodity crops[citation needed]. Recently, R&D has targeted enhancement of crops that are locally important in developing countries, such as insect-resistant cow-pea for Africa and insect-resistant Brinjal eggplant for India.
Transgenic plants have been used for bioremediation of contaminated soils. Mercury, selenium and organic pollutants such as polychlorinated biphenyls (PCBs) have been removed from soils by transgenic plants containing genes for bacterial enzymes.

Golden rice

Golden rice burst on a world ready and eager for a new beginning.
Announced with a flourish in January 2000, it promised to save millions of people from blindness and disease.
It can certainly help to improve nutrition and health in many developing countries.
But, as the publicists' dust settled, it became clear that golden rice was never going to be a silver bullet.
It is a genetically modified (GM) strain of rice that has been engineered to produce beta-carotene.
That not only gives it its eponymous golden colour, but enables people eating it to produce vitamin A.
The World Health Organisation estimates about 250 million people globally are deficient in vitamin A, increasing their risk of blindness, immune problems and other serious conditions.
Improving on Nature
So golden rice sounded like a real answer to a genuine problem, especially since the biotech company responsible, Zeneca, said it would offer the seeds freely to farmers in poor countries.
The reality, though, appears a little more prosaic. For a start, the genes for beta-carotene are already present in conventional rice.
It is just that they do not work as well in the "natural" varieties as in the novel version.
Beyond that though, poorly-fed people are unlikely to be able to absorb beta-carotene even when they eat golden rice. To use it, they need a diverse diet, including green leafy vegetables.
But the sorts of vegetables people used to be able to find have declined in number as the green revolution of the 60s and 70s emphasised monocultures of new varieties.
Household consumption of vegetables in India has fallen by 12% in two decades.
The prospects for golden rice receded a little further in 2002, when scientists published the draft sequences of the rice genome.
Short cut
That promised quicker results from conventional plant breeding, partly because it established where the beta-carotene "pathway" sat in the rice code.
A scientist from the biotech company Syngenta, which now includes Zeneca, said: "All the genes are present in rice. One could make a non-GM vitamin-A rice simply by studying those genes in a more focused way."
Golden rice may prove part of the answer to vitamin A deficiency, though not the comprehensive solution it seemed to be.
But it would be an answer that came with a hefty price-tag: the persistent concerns about the safety of GM technology to human health and to wild species.
Golden rice looks like being a special case, anyway, because the biotech industry is unlikely to give poor farmers free access to all its inventions.
People who campaign against GM crops are sometimes accused of wanting to deny the wretched of the Earth the chance to escape poverty and disease, all in the name of their own ideological obsession. But some impressive figures echo their concerns.
False dawn
Dr Richard Horton, editor of the British medical journal The Lancet, said: "Seeking a technological food fix for world hunger may be... the most commercially malevolent wild goose chase of the new century."
And from the biotech industry itself, Steve Smith, who worked for Syngenta Seeds before his death in June 2003, said: "If anyone tells you that GM is going to feed the world, tell them that it is not... To feed the world takes political and financial will - it's not about production and distribution."
Every day 800 million people go to bed with empty stomachs. Every day more than 30,000 under-fives die, from easily prevented diseases or from hunger.
The world is out of joint, and it will stay that way until those of us who are well-fed care enough to wage a war on hunger as ferocious as that against terrorism.
Science, perhaps including GM technology, can provide the weapons for that war - but that won't ever be a silver bullet.
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