AMITY INSTITUTE OF BIOTECHNOLOGY

ASSIGNMENT

ON

EMERGING TRANSGENICS IN GENETIC ENGINEERING

SUBMITTED BY

PRIYANKA AGGARWAL IMT/08/9056

PRIYANKA GOYAL IMT/08/9057

SECTION – I

SEMESTER 8TH

INTEGRATED M-TECH. BIOTECHNOLOGY

CONTENTS

What is Transgenic? Historical Background How are Transgenic Animals produced? Examples for Transgenic Animals How do Transgenic Animals contribute to

Human Welfare? What are the Ethical Concerns surrounding

Transgenesis?

LIST OF FIGURES

1. Methods for producing a transgenic animal2. Microinjection of DNA into a Pronucleus3. Retrovirus mediated gene transfer4. Embyronic Stem cell mediated gene transfer

WHAT IS TRANSGENICS?

In simple terms transgenic is the transfer of specific genes from one organism to another by the process of gene splicing which requires a vector for the transfer of the desired gene from the host to the recipient.

The main objectives of gene transfer includes the study on promoter function, reporter gene expression, regulation of gene expression, functions of transferred genes/DNA sequences, improve the production of milk, meat, wool, and also gene therapy.

Gene therapy is the insertion of genes into an individual’s cells and tissues to treat diseases especially hereditary diseases. It does so by replacing a defective mutant allele with a functional one or targeting which involves gene amplification. Transgenics have been produced in a variety of animal species, e.g. mice (including rats), rabbits, swine, goat, cattle, sheep, poultry, fish, amphibians, insects and nematodes.

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 the gene itself, 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

The Federation of European Laboratory Animal Associations defines the term as an animal in which there has been a deliberate modification of its genome, the genetic makeup of an organism responsible for inherited characteristics.[1]

The nucleus of all cells in every living organism contains genes made up of DNA. These genes store information that regulates how our bodies form and function. Genes can be altered artificially, so that some characteristics of an animal are changed. For example, an embryo can have an extra, functioning gene from another source artificially introduced into it, or a gene introduced which can knock out the functioning of another particular gene in the embryo. Animals that have their DNA manipulated in this way are knows as transgenic animals.[2]

The majority of transgenic animals produced so far are mice, the animal that pioneered the technology. The first successful transgenic animal was a mouse.[3] A few years later, it was followed by rabbits, pigs, sheep, and cattle.[4,5,6,7]

Why are these animals being produced?

Some transgenic animals are produced for specific economic traits. For example, transgenic cattle were created to produce milk containing particular human proteins, which may help in the treatment of human emphysema.

Other transgenic animals are produced as disease models (animals genetically manipulated to exhibit disease symptoms so that effective treatment can be studied). For example, Harvard scientists made a major scientific breakthrough when they received a U.S. patent (the company DuPont holds exclusive rights to its use) for a genetically engineered mouse, called OncoMouse® or the Harvard mouse, carrying a gene that promotes the development of various human cancers.[8]

HISTORICAL BACKGROUNDPrior to the development of molecular genetics, the only way of studying the regulation and function of mammalian genes was through the observation of inherited characteristics or spontaneous mutations. Long before Mendel and any molecular genetic knowledge, selective breeding was a common practice among farmers for the enhancement of chosen traits, e.g., increased milk production.

During the 1970s, the first chimeric mice were produced (Brinster, 1974). The cells of two different embryos of different strains were combined together at an early stage of development (eight cells) to form a single embryo that subsequently developed into a chimeric adult, exhibiting characteristics of each strain.

The mutual contributions of developmental biology and genetic engineering permitted rapid development of the techniques for the creation of transgenic animals. DNA microinjection, the first technique to prove successful in mammals, was first applied to mice (Gordon and Ruddle, 1981) and then to various other species such as rats, rabbits, sheep, pigs, birds, and fish. Two other main techniques were then developed: those of retrovirus-mediated transgenesis (Jaenisch, 1976) and embryonic stem (ES) cell-mediated gene transfer (Gossler et al., 1986).

Since 1981, when the term transgenic was first used by J.W. Gordon and F.H. Ruddle (1981), there has been rapid development in the use of genetically engineered animals as investigators have found an increasing number of applications for the technology.

HOW ARE TRANSGENIC ANIMALS PRODUCED?

Since the discovery of the molecular structure of DNA by Watson and Crick in 1953, molecular biology research has gained momentum. Molecular biology technology combines techniques and expertise from biochemistry, genetics, cell biology, developmental biology, and microbiology.[9]

Scientists can now produce transgenic animals because, since Watson and Crick’s discovery, there have been breakthroughs in:

recombinant DNA (artificially-produced DNA) genetic cloning analysis of gene expression (the process by which a gene gives rise to a

protein) genomic mapping

The underlying principle in the production of transgenic animals is the introduction of a foreign gene or genes into an animal (the inserted genes are called transgenes). The foreign genes “must be transmitted through the germ line, so that every cell, including germ cells, of the animal contain the same modified genetic material”26 (Germ cells are cells whose function is to transmit genes to an organism’s offspring.)

To date, there are three basic methods of producing transgenic animals:

DNA microinjection Retrovirus-mediated gene transfer Embryonic stem cell-mediated gene transfer

Gene transfer by microinjection is the predominant method used to produce transgenic farm animals. Since the insertion of DNA results in a random process, transgenic animals are mated to ensure that their offspring acquire the desired transgene. However, the success rate of producing transgenic animals individually by these methods is very low and it may be more efficient to use cloning techniques to increase their numbers. For example, gene transfer studies revealed that only 0.6% of transgenic pigs were born with a desired gene after 7,000 eggs were injected with a specific transgene.[10]

Figure 1 – Methods for producing a transgenic animal

1. DNA MICROINJECTION

The mouse was the first animal to undergo successful gene transfer using DNA microinjection.[2] This method involves:

transfer of a desired gene construct (of a single gene or a combination of genes that are recombined and then cloned) from another member of the same species or from a different species into the pronucleus of a reproductive cell[11]

the manipulated cell, which first must be cultured in vitro (in a lab, not in a live animal) to develop to a specific embryonic phase, is then transferred to the recipient female.

Figure 2 – Microinjection of DNA into a Pronucleus

2. RETROVIRUS-MEDIATED GENE TRANSFER

A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. This method involves[12] :

retroviruses used as vectors to transfer genetic material into the host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic constitution

chimeras are inbred for as many as 20 generations until homozygous (carrying the desired transgene in every cell) transgenic offspring are born

The method was successfully used in 1974 when a simian virus was inserted into mice embryos, resulting in mice carrying this DNA.[13]

Figure 3 – Retrovirus mediated gene transfer

3. EMBRYONIC STEM CELL-MEDIATED GENE TRANSFER

This method involves:[14,11,12]

isolation of totipotent stem cells (stem cells that can develop into any type of specialized cell) from embryos

the desired gene is inserted into these cells cells containing the desired DNA are incorporated into the host’s embryo,

resulting in a chimeric animal

Unlike the other two methods, which require live transgenic offspring to test for the presence of the desired transgene, this method allows testing for transgenes at the cell stage.

Figure 4 – ES cell mediated gene transfer

EXAMPLES FOR TRANSGENIC ANIMALS

Transgenic animals are used as experimental models to perform phenotypic and for testing in biomedical research.[15]

Genetically-Modified (Genetically Engineered) animals are becoming more vital to the discovery and development of cures and treatments for many serious diseases. By altering the DNA or transferring DNA to an animal, we can develop certain proteins that may be used in medical treatment. Stable expressions of human proteins have been developed in many animals, including sheep, pigs, and rats.

Some examples are: Human-alpha-1-antitrypsin,[16] which has been developed in sheep and is used in treating humans with this deficiency and transgenic pigs with human histo-compatibility have been studied in the hopes that the organs will be suitable for transplant with less chances of rejection. Transgenic livestock have been used as bioreactors since the 1990s. Many medicines, including insulin and many immunizations are developed in transgenic animals.[17] In March 2011, the bioactive recombinant Human Lysozyme was expressed in the milk of cloned transgenic cattle. This field is growing rapidly and new pharming uses are being discovered and developed. The extent that transgenic animals will be useful in the medical field as well as other fields is very promising based on results thus far.[18]

Fruit flies

In biological research, transgenic fruit flies (Drosophila melanogaster) are model organisms used to study the effects of genetic changes on development.[19] Fruit flies are often preferred over other animals due to their short life cycle, low maintenance requirements, and relatively simple genome compared to many vertebrates.

Mosquitoes

In 2010, scientists created "malaria-resistant mosquitoes" in the laboratory.[20,21,22] The World Health Organisation estimated that Malaria killed almost one million people in 2008.[23] Genetically modified male mosquitoes containing a lethal gene have been developed in order to combat the spread of Dengue fever.[24] Aedes aegypti mosquitoes, the single most important carrier of dengue fever, were reduced by 80% in a 2010 trial of these GM mosquitoes in the Cayman Islands.[25]

Between 50 - 100 million people are affected by Dengue fever every year and 40,000 people die from it.[26]

Bollworms

A strain of Pectinophora gossypiella (Pink bollworm) has been developed that contains a fluorescent marker in their DNA. This allows researchers to monitor bollworms that have been sterilized by radiation and released in order to reduce bollworm infestation.[26,27]

Mammals

Genetically modified mammals are an important category of genetically modified organisms. Transgenic mice are often used to study cellular and tissue-specific responses to disease.

In 1999, scientists at the University of Guelph in Ontario, Canada, created the genetically engineered Enviropig. The Enviropig excretes from 30 to 70.7% less phosphorus in manure depending upon the age and diet.[28] In February 2010, Environment Canada determined that Enviropigs are in compliance with the Canadian Environmental Protection Act and can be produced outside of the research context in controlled facilities where they are segregated from other animals.[29]

In 2009, scientists in Japan announced that they had successfully transferred a gene into a primate species (marmosets) and produced a stable line of breeding transgenic primates for the first time.[30,31] Their first research target for these marmosets was Parkinson's disease, but they were also considering Amyotrophic lateral sclerosis and Huntington's disease.[32]

In 2011, scientists in China released news that they have introduced human genes into 300 dairy cows to produce milk with the same properties as human breast milk. Aside from milk production, the researchers claim these transgenic cows to be identical to regular cows.[33]

Cnidarians

Cnidarians such as Hydra and the sea anemone Nematostella vectensis have become attractive model organisms to study the evolution of immunity and certain developmental processes. An important technical breakthrough was the

development of procedures for generation of stably transgenic hydras and sea anemones by embryo microinjection.[34]

Fish

Genetically modified fish have promoters driving an over-production of "all fish" growth hormone. This resulted in dramatic growth enhancement in several species, including salmonids,[35] carps[36] and tilapias.[37]

Transgenic Chickens

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 carryingo the human gene for a therapeutic proteino 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.

Transgenic Primates

In the 28 May 2009 issue of Nature, Japanese scientists reported 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.

HOW DO TRANSGENIC ANIMALS CONTRIBUTE TO HUMAN

WELFARE?The benefits of these animals to human welfare can be grouped into areas:

Agriculture Medicine Industry

The examples below are not intended to be complete but only to provide a sampling of the benefits.

1. Agricultural Applications

a) Breeding Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate). Traditional breeding is a time-consuming, difficult task. When technology using molecular biology was developed, it became possible to develop traits in animals in a shorter time and with more precision. In addition, it offers the farmer an easy way to increase yields.

b) Quality Transgenic cows exist that produce more milk or milk with less lactose or cholesterol12, pigs and cattle that have more meat on them , and sheep that grow more wool. In the past, farmers used growth hormones to spur the development of animals but this technique was problematic, especially since residue of the hormones remained in the animal product.

c) Disease resistance Scientists are attempting to produce disease-resistant animals, such as influenza-resistant pigs, but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals.

2. Medical Applications

a) Xeno-transplantation Patients die every year for lack of a replacement heart, liver, or kidney. For example, about 5,000 organs are needed each year in the United Kingdom alone. Transgenic pigs may provide the transplant organs needed to alleviate the shortfall. Currently xeno-transplantation is hampered by a pig protein that can cause donor rejection but research is underway to remove the pig protein and replace it with a human protein.

b) Nutritional supplements and pharmaceuticals Products such as insulin, growth hormone, and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows, sheep, or goats. Research is also underway to manufacture milk through transgenesis for treatment of debilitating diseases such as phenylketonuria (PKU), hereditary emphysema, and cystic fibrosis.

In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk at 2.4 grams per litre. This transgenic milk is a more nutritionally balanced product than natural bovine milk and could be given to babies or the elderly with special nutritional or digestive needs. Rosie’s milk contains the human gene alpha-lactalbumin.

c) Human gene therapy Human gene therapy involves adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene. The potential for treatments for the 5,000 named genetic diseases is huge and transgenic animals could play a role. For example, the A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans.

3. Industrial Applications

In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. The goats began to manufacture silk along with their milk and secrete tiny silk strands from their body by the bucketful. By extracting polymer strands from the milk and weaving them into thread, the

scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings.

Toxicity-sensitive transgenic animals have been produced for chemical safety testing. Microorganisms have been engineered to produce a wide variety of proteins, which in turn can produce enzymes that can speed up industrial chemical reactions.

WHAT ARE THE ETHICAL CONCERNS SURROUNDING

TRANSGENESIS?This article focuses on the benefits of the technology; however, thoughtful ethical decision-making cannot be ignored by the biotechnology industry, scientists, policy-makers, and the public. These ethical issues, better served in their own article, include questions such as:

Should there be universal protocols for transgenesis?

Should such protocols demand that only the most promising research be permitted?

Is human welfare the only consideration? What about the welfare of other life forms?

Should scientists focus on in vitro (cultured in a lab) transgenic methods rather than, or before, using live animals to alleviate animal suffering?

Will transgenic animals radically change the direction of evolution, which may result in drastic consequences for nature and humans alike?

Should patents be allowed on transgenic animals, which may hamper the free exchange of scientific research?

CONCLUSIONTransgenic animals can be produced and manufactured to serve a variety offunctions previously unheard of. Genetically modified animals have evolved into easy tools for scientists to use that could very well become as commonly used as laboratories or factories.Since the early eighties, research as shifted from focusing on how to maketransgenic animals to what industries these animals can be applied to produce maximum benefits. What began with a mouse, has progressed into five major categories encompassing all different types of microorganisms. These groups include disease models, transpharmers, xenoplanters, food sources, and scientific models. Disease models such as the Alzheimer’s Mouse, the Oncomouse, and the AIDs mouse are individual lab models used to speed up the research in developing cures and treatments for specific diseases. Dairy cattle, goats, and sheep make up the transpharmers group. These animals act as bioreactor factories to produce pharmaceuticals. Transpharmers are ideal tools for manufacturing cheap drugs, without any expense to the animals.Xenotransplanters, include genetically modified pigs with organs that can be donated to human beings. In the event that organs from pigs can be successfully transplanted to humans, patients waiting for organs will become a thing of the past, and hospitals will save an uncountable amount of money in operating costs. The next category is food sources; currently the largest impact on society in this category has been the modification of salmon to produce bigger, meater fish. Chickens are also in this category, because researchers are trying to create disease resistant models that would bring out obvious benefits to both the consumer and producer. Finally, there are scientific models that bring about a better understanding of how specific proteins work in vivo.

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