Transgenic Animals-A Boon By Biotechnology

Neha Vithani

Defination And Introduction To The Transgenic System:

Nowadays, breakthroughs in molecular biology are happening at an unprecedented rate. One of them is the ability to engineer transgenic animals. The term "transgenics" refers to the science of inserting a foreign gene into an organism's genome. An animal is "transgenic" once a scientist inserts DNA from another organism. This process allows scientists to transfer beneficial genes from a different animal, bacterium, or plant. ^[6]

Defining The Term - Transgenic Animal:

There are various definitions for the term transgenic animal.

A transgenic animal is one whose genome has been changed to carry genes from other species^{. [1]}

Transgenic animals are animals which have been genetically transformed by splicing and inserting foreign animal or human genes into their chromosomes.

The term transgenic animal refers to an animal in which there has been a deliberate modification of the genome - the material responsible for inherited characteristics - in contrast to spontaneous mutation. ^[2]

A transgenic animal is one which has been genetically altered to have specific characteristics it otherwise would not have. In animals, transgenesis either means transferring DNA into the animal or altering DNA already in the animal.

Transgenic animals contain elements of two different species - they are creatures that blur the barrier between species.

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]

An Introduction To The Technique:

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. ^[1]

Transgenic animals are produced by inserting genes into embryos prior to birth. Each transferred gene is assimilated by the genetic material or chromosomes of the embryo and subsequently can be expressed in all tissues of the resulting animal. The objective is to produce animals which possess the transferred gene in their germ cells (sperm or ova). Such animals are able to act as "founder" stock to produce many offspring that carry a desirable gene or genes. The animal that develops after receiving the transgene DNA is referred to as the founder (Fo) of a new transgenic lineage. If the germ cells of the founder (mosaic or not) transmit the transgene stably, then all descendants of this animal are members of a unique transgenic lineage.

A transgenic animal carries heterologous DNA stably integrated into its genome. A transgenic animal results from insertion of a foreign gene into an embryo. The foreign gene becomes a permanent part of the host animals' genetic material. As the embryo develops, the foreign gene may be present in many cells of the body, including the germ cells of the testis or the ovary. If the transgenic animal is fertile, the inserted foreign gene (transgene) will be inherited by future progeny. Thus, a transgenic animal, once created, can persist into future generations.

Transgenic animals are different from animals in which foreign cells or foreign organs have been engrafted. The progeny of engrafted animals do not inherit the experimental change. The progeny of transgenic animals do.

Why Transgenic Animals?

In some cases over expression of human genes in bacteria (such as E. coli) does not yield a protein that is functionally active in humans. The reason for this is that some proteins need to be post-translationally modified (phosphorylated, glycosylated, etc.) before they are active. Bacteria generally lack the specific enzymes recognizing the human protein sequences that need to be modified, and thus the bacterially produced gene product will differ from the native one. To counter this problem, certain human genes can be introduced into farm animals (usually yeast will do the job, too), and when these genes are expressed in the mammary glands of the animals, the post-translationally modified protein can be isolated from milk, tested whether its post-translationally modified product is identical or at least very similar to the native human one, and if so, be developed as a pharmaceutical. For example, the genes for two different human blood clotting factors (VIII and IX) have been hooked up to sheep and pig regulatory sequences that causes expression in mammary tissue; after transformation of sheep or pig embryos, genetically engineered animals have been selected that produce milk with a large percentage of human blood-clotting factor. This protein can be isolated from the milk, purified, and marketed. Similarly, transgenic rabbits have been created that produce human interleukin-2, which is a protein stimulating the proliferation of T-lymphocytes; the latter play an important role in fighting selected cancers. ^[3]

The majority of transgenic animals produced so far are mice, the animal that pioneered the technology. Long life cycles of farm animals slow genetic analysis. That's why researchers use smaller, faster-breeding animals such as mice as model systems to test their ideas and their DNA constructs. The first successful transgenic animal was a mouse. Over 80% of mouse genes function the same as those in humans. Mice also have a short reproduction cycle and their embryos are amenable to manipulation. Mice are therefore an ideal human surrogate in the study of most diseases. Currently over 95% of transgenic animals used in biomedical research are mice. Other transgenic animals include rats, pigs and sheep. It is hoped that the refinement of transgenesis techniques in mice will ultimately allow for a corresponding reduction in the use of "higher" animals, such as dogs and non-human primates, in biomedical research.A few years later; it was followed by rabbits, pigs, sheep, fish, poultry and cattle. Furthermore, the mouse is the only model system that combines homologous recombination with cloning to allow the study of modified genes in development of adult animals. Currently, in livestock homologous recombination is possible only with cells grown in tissue culture. This means a scientist can study the effect of knocked-out genes only on the physiology of the cell. The possible role of the gene in development from embryo to adult cannot be tested without a system of cloning: taking the original cell and growing an adult from it.

The insertion of a foreign gene (transgene) into an animal is successful only if the gene is inherited by offspring. The success rate for transgenesis is very low and successful transgenic animals need to be cloned or mated.

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.

Scientists do this, creating a "transgenic" organism, to study the function of the introduced gene and to identify genetic elements that determine which tissue and at what stage of an organism's development a gene is normally turned on. Transgenic animals have also been created to produce large quantities of useful proteins and to model human disease. Various human proteins that have been expressed in transgenic animals include: anti-thrombin III (to treat intravascular coagulation), collagen (to treat burns and bone fractures), fibrinogen (used for burns and after surgery), human fertility hormones, human hemoglobin, human serum albumin (for surgery, trauma, and burns), lactoferrin (found in mother milk), tissue plasminogen activator, and particular monoclonal antibodies (including one that is effective against a particular colon cancer). Animals mostly used for this work are pigs, cows, sheep, and goats.

Almost all the work on transgenic animals is still at the research level. But it enjoys inherent interest and the immense potential for future commercial applications. Scientists, farmers and business corporations hope that transgenic techniques will allow more precise and cost-effective animal and plant breeding programs. They also hope to use these new methods to produce animals with desirable characteristics that are not available using current breeding technology.

The technology has already produced transgenic animals such as mice, rats, rabbits, pigs, sheep, and cows. Although there are many ethical issues surrounding transgenesis, this article focuses on the basics of the technology and its applications in agriculture, medicine, and industry.

Historical Background

Prior 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. ^[2]

In the 1970s, experiments were conducted with embryonal carcinoma cells and teratocarcinoma cells to construct chimeric mice (Brinster, 1974; Mintz and Illmensee, 1975; Bradley et al., 1984). In these chimeric animals, cultured cells derived from one strain of mouse were introduced into the embryos of another strain of mouse by direct embryo aggregation or by injection into the blastocyst stage embryo. ^[2]

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). ^[2]

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. ^[2]

During 1982-83 pioneering research featured transgenic mice given a copy of the gene for human growth hormone. In this case, the added gene was inserted randomly in the mouse genome. It did not insert at the mouse gene for growth hormone. The addition of the gene for human growth hormone did not inactivate or "knock-out" the genes for mouse growth hormone. ^[4]

A picture of two mice side by side, one with the extra gene and the other without, gave "visual impact of what this technology might do," Pinkert pointed out. Comparing the size of the mouse with the extra gene to the control was like comparing a softball to a baseball. ^[4]

In 1997 the cloning of Dolly and engineering of Polly have combined transgenesis and cloning. This combination was an essential step in developing in livestock a system of homologous recombination to modify existing genes. ^[4]

Timeline Of Animal Cloning And Gene Transfer:

·1891 first successful embryo transfer early 1900's in vitro embryo culture develops

· 1961 mouse embryo aggregation to produce chimeras

· 1966 first report of microinjection of mouse embryos

· 1973 foreign genes function after cell transfection

· 1974 development of teratocarcinoma cell transfer

· 1977 mRNA and DNA transferred to Xenopus eggs

· 1980 mRNA transferred into mammalian ova

· 1980-81 transgenic mice first documented

· 1981 transfer of ES cells derived from mouse embryos

· 1982 transgenic mice and a growth hormone phenotype

· 1983 tissue specific gene expression in transgenic mice

· 1985 transgenic domestic animals produced

· 1985 microinjection for transgenic pigs, sheep, rabbits, fish

· 1987 chimeric "knock-out" mice described

· 1987 retrovirus mediated: transgenic chicken

· 1989 targeted DNA integration & germline chimeric mice

· 1989 microinjection for transgenic cattle ( Russia )

· 1989 first sperm mediated reports in farm animals

· 1991 microinjection for transgenic goats first refereed publication

· 1993 germline chimeric mice produced using co-culture

· 1996 ES cells used for nuclear transfer: sheep

· 1997 somatic cells from adult sheep used for cloning by nuclear transfer (Dolly). ^[4]

Production Of Transgenic Animals:

Scientists can now produce transgenic animals because, since Watson and Crick’s discovery (in 1953), there have been breakthroughs in:

1. Recombinant DNA (artificially-produced DNA)

2. Genetic cloning

3. Analysis of gene expression (the process by which a gene gives rise to a protein)

4. Genomic mapping ^[1]

Principle:

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 contains the same modified genetic material.”(Germ cells are cells whose function is to transmit genes to an organism’s offspring.)^[1]

This is a brief outline of the steps necessary to obtain transgenic mice or rats: DNA is prepared and microinjected into fertilized mouse or rat eggs. Potentially transgenic rodents are born. Transgenic founders are identified and bred to produce offspring for analysis. Core personnel are available for consultation on all aspects of transgenic research. Much genetic engineering goes into the choice of a foreign gene and building a construct. The construct must have promotes to turn on foreign gene expression at its new site within the host animal genome. By choosing a particular promoter and splicing it in front of the foreign gene, we can encourage expression of our transgene within a specific tissue.

A transgenic animal for pharmaceutical production should (1) produce the desired drug at high levels without endangering its own health and (2) pass its ability to produce the drug at high levels to its offspring. The current strategy to achieve these objectives is to couple the DNA gene for the protein drug with a DNA signal directing production in the mammary gland. The new gene, while present in every cell of the animal, functions only in the mammary gland so the protein drug is made only in the milk. Since the mammary gland and milk are essentially "outside" the main life support systems of the animal, there is virtually no danger of disease or harm to the animal in making the "foreign" protein drug. After the DNA gene for the protein drug has been coupled with the mammary directing signal, this DNA is injected into fertilized cow, sheep, goat, or mouse embryos with the aid of a very fine needle, a tool called a micromanipulator, and a microscope (Figure 2). The injected embryos are then implanted into recipient surrogate mothers where, hopefully, they survive and are born normally. ^[14]

Production of Transgenic animal is entirely described here:

1. Plan the experiment:

What is the purpose of your experiment? Do you want to define tissue specific regulatory sequences? Do you want to overexpress a protein in a specific cell lineage? You will need to obtain or clone the desired promoter and structural gene. Expression of some genes will be deleterious or incompatible with proper growth and development of the embryo. Special arrangements should be made if you expect embryonic lethality from transgene expression. The expression of a transgene requires that the appropriate transcriptional control elements be included in the DNA construct. A literature search may identify these elements. Preliminary studies in cell cultures are recommended to verify the integrity of the construct and the function of the promoter. However, it is not always possible to predict in advance whether the transgene will have the capability of being expressed in vivo. A review of reporter molecules is available. The Core has a nuclear localized lacZ reporter vector for investigators who wish to characterize regulatory elements in transgenic mice. A completed materials transfer agreement is required before this plasmid can be distributed to investigators. Commercially available vectors that may be useful in transgenic research include: 1) the CMV-IE promoter for widespread gene expression, 2) tetracycline regulated gene expression systems for inducible gene expression, and 3) luciferase and green fluorescent protein reporter genes. ^[7]

2. Transgene DNA Preparation :

Although the basic coding system is the same in all organisms, the fine details of gene control often differ. A gene from a bacterium, say, will often not work correctly if it is introduced unmodified into a plant or animal cell. The genetic engineer must first construct a transgene – the gene to be introduced plus a control sequence. All genes are controlled by a special segment of DNA found on the chromosome next to the gene and called a promoter sequence. When constructing a transgene, scientists generally substitute the original promoter sequence with one that will be active in the correct tissues of the recipient animal or plant and which may also allow them to turn the gene on or off as needed. For example, a promoter sequence that requires a dietary "trigger" substance can be used to turn on a new hormone gene in animals; the animal does not produce the new hormone unless fed the appropriate trigger.

In order for a transgenic technique to work, the genetic engineer must first construct a transgene, which is the gene to be introduced plus a control sequence. When making a transgene, scientists usually substitute the original promoter sequence with one that will be active in the correct tissues of the recipient plant or animal. The gene for the target protein is linked with a milk-specific promoter. Simply put, the investigator constructs a transgene with a promoter and a structural gene for example a reporter gene such as lacZ or a transcription factor. ^[9]

The transgene DNA is engineered in the molecular laboratory to achieve fairly predictable expression in the animal. Using restriction enzymes and ligase, different functional regions of genes from different species may be recombined in the test tube. All components of endogenous genes may be isolated and recombined to form a transgene expression cassette or construct. The ends of the completed construct may be modified by the addition of polylinker sequences containing several different restriction enzyme recognition sites. The polylinker permits the construct to be inserted into a variety of vectors for testing and cloning. The following review of endogenous gene components will clarify these strategies:

(Figure 1. Schematic diagram of the double-stranded DNA regions of the transgene expression cassette. )

Restriction enzyme recognition sites are clustered at either end of the cassette (i.e., upstream and downstream).
"ATG" indicates the beginning of the transcriptional reading frame.
"SIG" indicates the signal sequence.
"AAA" indicates the poly-A tail ^[9]

The endogenous gene contains exons that code for specific portions of the final protein and introns that appear necessary for optimal expression of the gene (Figure 2). The endogenous gene is flanked by non-coding DNA sequences that regulate gene expression. (Regulatory elements may also reside within intragenic intron sequences.) Sequences located at the 5' end of the gene are known as upstream elements, while downstream elements are found past the 3' end of the gene sequence. Regulatory elements called promoters are usually found immediately upstream of the gene, and have critical roles in the temporal and tissue-specific regulation of gene expression. Other regulatory elements called enhancers function to enhance gene expression, independent of their location and orientation with respect to the gene. Enhancer regions appear to correlate with DNAase hypersensitive sites and may be several kilo bases (kb) distant from the gene. Signal sequences are short sequences that target protein synthesis into specific intracellular pathways and frequently direct secretion of the protein from the cell. Secretory signals usually are found directly adjacent to the 5' end of the gene, and organelle targeting sequences usually are found within the 3' end of the gene or immediately downstream of the 3' end. These signal sequences are within the reading frame or transcriptional region of the gene and therefore encode mRNA and short polypeptide products. The 3' end of the reading frame also must contain a poly-A nucleotide sequence to ensure proper mRNA transcription and translation. ^[9]

(Figure 2. Comparison of the two forms of transgenes that may be introduced into embryonic pronuclei.)

The genomic form includes all naturally-occurring intron elements that are involved in mRNA splicing and expression, whereas the shorter cDNA form is a synthetic sequence representing only the protein-encoding exon elements of the gene. ^[9]

Steps of transgene preparation:

1) The gene of interest is isolated on a strand of DNA.

2) DNA is cut at specific points by restriction enzymes. The enzymes recognize certain sequences of bases on the DNA strand and cut where those sequences appear.

3) The cut DNA joins with a vector, which may be a virus or part of a bacterial cell called a plasmid. The vector carries the gene of interest into the organism that will produce the protein.

4) Transformation occurs when the gene carried by the vector is incorporated into the DNA of another organism where it initiates the action desired (production of a drug, etc.) ^[14]

3. Insertion of the transgene into animals :

By genetic engineering, the DNA gene for a protein drug of interest can be transferred into another organism that will produce large amounts of the drug. This technique can be used to impart new production characteristics to an organism, as well as to trigger the production of a protein drug. Copies of the transgene are usually injected directly into a fertilized egg which is then implanted in the female productive tract. However, it is difficult to control where in the chromosome the transgene is inserted, and this sometimes causes variations in the level at which the gene is expressed. As well, the process is technically demanding and has a low success rate. Currently less than 5 per cent of injected embryos result in offspring with the gene integrated into their DNA and able to be passed on consistently to successive generations. Researchers are therefore investigating new methods of gene transfer

Gene Transfer Methods:

1.Microinjection of cells (oocytes) with DNA

2.ES (embryonic stem) cell transfer

3.Retroviral infection

4.Blastomere/embryo aggregation

5.Teratocarcinoma cell transfer

6.Electrofusion

7.Nuclear transplantation

8.Sperm-mediated transfer

9.Particle bombardment ("gene gun")

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

· DNA microinjection

· Retrovirus-mediated gene transfer

· Embryonic stem cell-mediated gene transfer^[1]

(A). DNA Microinjection:

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. The mouse was the first animal to undergo successful gene transfer using DNA microinjection. ^[1]

(Figure 3. Sequence of events in the generation of a transgenic animal by pronuclear microinjection) ^[1]

A. The double-stranded DNA components of the transgene are combined enzymatically to yield a transgene expression cassette.
B. Transgene cassettes are inserted into plasmid vectors and cloned.
C. Transgene-bearing plasmids are transfected into cultured eukaryotic cells to evaluate expression of the transgene.
D. Plasmid-free transgene fragments are introduced directly into embryonic pronuclei.
E. Manipulated embryos are placed in the reproductive tract of a pseudopregnant recipient.
F. The genomic DNA of live-born pups is analyzed for the presence of the transgene DNA sequence. ^[9]

(Injection of cloned DNA into embryos: One cell embryo is positioned for micro-injection into the pronucleus (left). The plasma membrane has been pierced, and the tip of the needle remains inside the pronucleus, while DNA is expelled from the needle, causing the pronucleus to swell visibly.)

This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum. It is one of the first methods that proved to be effective in mammals (Gordon and Ruddle, 1981). The introduced DNA may lead to the over- or under-expression of certain genes or to the expression of genes entirely new to the animal species. The insertion of DNA is, however, a random process, and there is a high probability that the introduced gene will not insert itself into a site on the host DNA that will permit its expression. The manipulated fertilized ovum is transferred into the oviduct of a recipient female or foster mother that has been induced to act as a recipient by mating with a vasectomized male.

The first successful production of transgenic mice using pronuclear microinjection was reported in 1980 (Gordon et al., 1980)

Embryo Collection:

The choice of the donor parental strains for production of the pronuclear embryos is a point of extreme proprietary concern to most laboratories. Many factors are cited including the response to superovulation, frequencies of embryo survival following microinjection, size of pronuclei and the incidence of specific pathologies inherent in various strains. The relative merit of inbred versus outbred backgrounds may be important for the evaluation of a specific transgene expression. Other factors may involve coat color, the availability of a certain strain, or simply anecdotal rationales ^[9]

On the morning following breeding, oviducts are removed from euthanized donors, and clumps of pronuclear embryos are collected from the oviducts by flushing or by dissection into a microdrop of sterilized buffered medium. The embryos are clumped together with sticky follicular cumulus cells that must be removed by brief treatment in a series of microdrops. The first drop, a solution of the enzyme hyaluronidase, is followed by two or more wash drops. Using heat-pulled tapered micropipettes controlled by mouth suction (a new pipette for each drop), the embryos are transferred from drop to drop until they are free of cumulus cells, debris and enzyme. Finally, the embryos are transferred into a pool of medium in a petri dish that will be placed under the microscope. The embryo-containing pool is covered by a layer of sterile-filtered, autoclaved mineral oil to prevent contamination by microorganisms and debris and to prohibit evaporation and the resultant pH changes that would kill the embryos. All collection and manipulation media contain a buffering system (i.e., bicarbonate or HEPES) and protein source (e.g., bovine serum albumin) to prevent embryos from adhering to the dishes and pipettes. In addition, media may contain antibiotics (e.g., penicillin and/or streptomycin) and a heavy-metal chelating agent (e.g., EDTA).

Equipment:

The equipment required to perform microinjection can cost between $50,000 and $80,000 and includes:

·CO2 incubator to maintain manipulated embryos at 37-38° C in an atmosphere of 5-6 percent CO2.

·Inverted microscope with a fixed stage.

·Phase contrast, Nomarski differential interference, or Hoffman modulated contrast optical systems to visualize pronuclei. With 10x or 15x eyepieces, a 20x or 40 x objectives is required.

· A pair of micromanipulators to control the DNA injection pipette and the embryo-holding pipette.

· A pair of micro-volume syringes and associated tubing to regulate the fluid dynamics in the injection and holding pipettes. (Expensive automatic microinjection systems are available in lieu of the injection syringe.)

· Pipette-pulling apparatus.

· Vibration-free pneumatic table (optional).

· Microforge apparatus to heat-polish and bend pipets.

· Pipette bevelling apparatus (optional).

· Supply of clean capillary pipettes for the manufacture of holding and injection pipettes.

· Fluorinert solution (optional) to provide optimal fluid dynamics in the pipettes.

· Microphotographic equipment (optional) including 35mm camera and/or video recording apparatus. ^[9]

The petri dish containing the embryo microdrop is placed into focus at a relatively low magnification, and degenerated embryos may be culled from the healthy embryos at this time. The holding pipet is brought down into the medium, and the first embryo is gently sucked onto the end of the pipet and held in place. The tip of the injection pipet is brought into the same plane of focus as the pronucleus to be injected, and a small amount of DNA solution is ejected to ensure the patency of the pipet. The injection pipet is then thrust through the zona pellucida, cell membrane, cytoplasm and nuclear membrane in a single smooth motion. Even if the membrane appears to have been pierced, the only reliable indication of success is the swelling of the pronucleus (volume = approximately 1 pl). The pipet is removed smoothly, and the injected embryo is moved to the far end of the pool of medium before the next is processed. Once a group of embryos has been completed, it is transferred in a single volume of medium to another dish for incubation and visual evaluation within a few hours. All apparently viable embryos are then transferred to a recipient female oviduct.

Embryo Transfer:

The manipulated embryos must be transferred into a suitable reproductive tract in order to have an opportunity to become live-born transgenic mice. The recipient female optimally should be somewhat earlier in her reproductive cycle than the embryo donor because manipulated and cultured embryos exhibit slightly retarded development when compared to embryos that developed in vivo. Recipients for embryo transfer are prepared by mating with vasectomized males at the same time that the superovulated donor females are mated with fertile males. Recipient females are anesthetized, the skin and peritoneum are incised, and the ovarian fat pad and bursa are exteriorized and draped over the midline. The bursa is opened, avoiding any prominent vessels, and the infundibulum is located. An embryo transfer pipet with an internal diameter of less than 150 µm is loaded in the following sequence: one small air bubble, approximately 10 µl of medium, a second air bubble, 2-15 embryos in less than 25 µm of medium, and a third air bubble. The pipet tip is inserted into the infundibulum of the oviduct, and the contents are gently transferred into the oviduct by mouth pressure until the middle air bubble is expelled. The reproductive tract is gently replaced and the incision is closed. Pregnancy should be visible about two weeks after the embryo transfer (post-ET), and the litter should be delivered about three weeks post-ET. Animals may be analyzed for the presence of the transgene in their genomes after weaning at six weeks post-ET (Figure 3F). It should be noted that certain transgene sequences may be activated in uterus and may affect embryo survival or gestation length. Also, transgenic females in subsequent generations should be observed for abnormal gestation lengths.

A major advantage of this method is its applicability to a wide variety of species.

(B). Embryonic stem cell-mediated gene transfer:

This method involves:

· 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. ^[1]

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. ^[1]

This method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method. ^[2]

This technique is of particular importance for the study of the genetic control of developmental processes. This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination. ^[2]

(C). Retrovirus-mediated gene transfer.

The third method produces chimeras, altered animals with mixed DNA. To increase the probability of expression, gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to transfer genetic material into the cell, taking advantage of their ability to infect host cells in this way. Offspring derived from this method are chimeric, i.e., not all cells carry the retrovirus. Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells. ^[2]

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

1. 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.

2. 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.

4. Selection of Gene Targeted Cells :

Homologous recombination is a very rare event, and scientists using it to modify or "knock out" mouse genes must identify the cells in which it has occurred. In addition to injecting the gene they are trying to incorporate, scientists also inject "selectable" genes whose products permit cells to live or cause them to die in the presence of a particular drug. The two most common selectable genes used in gene targeting are the neomycin resistance (neo^r) gene, which allows cells to survive in the presence of the antibiotic neomycin, G418, and the thymidine kinase (TK) gene from the herpes virus. Cells with this gene die in the presence of the antiviral agent gancyclovir. The neo^r and TK genes are generally used together for maximum selection. ^{[12, 15]}

Scoreable vs. Selectable Markers:

Scoreable markers are an example of genes that are easy to find. Examples include a gene that makes an enzyme that makes a colour (such as GUS) or that makes light (such as lux genes for luciferase) or that makes a fluorescent protein (such as the gene for Green Fluorescent Protein). The two genes the 'gene of interest' and the 'selectable marker' are attached ('in tandem'). So where the scoreable marker goes, it is very likely the gene of interest goes too.

Another example of a gene that is easy to find is a gene for resistance to an antibiotic. This is called a selectable marker. These are even more powerful than scoreable markers. Cells that receive a copy of the gene for resistance to the antibiotic kanamycin can grow in test tubes containing kanamycin, while other cells without the gene will die.

With a scoreable marker, you may be able to pick out the one fluorescent cell in a thousand other cells that don’t fluoresce. But with a selectable marker, only the cells with the marker remain alive after treating with the antibiotic. It is the difference between an orange vest to make a cell stand out in a crowd and a bullet-proof vest that lets a cell survive the attack of antibiotics. ^[12]

Now you have your gene of interest and you can follow it using the attached selectable marker.

5. Clone and the Verify the Integrity of the Transgene :

In transgene design several things should be considered during cloning. For exempt, prokaryotic vector sequences interfere with the expression of some transgenes, thus unique restriction sites at the 5' and 3' ends of the construct should be available for vector removal. The transgene should contain unique markers so that its presence can be easily detected in DNA samples and so that its expression can be assayed and distinguished from endogenous gene expression. Sequencing of junction fragments should be carried out in order to confirm that the transgene has a functional promoter, initiation codon, and polyadenylation signal. Under the best circumstances, the transgene is tested for expression in a tissue culture system before transgenic mice are made. ^[7]

6. DNA Purification and MICROINJECTION

7. Establish a Screening Method :

A PCR assays should be established to rapidly identify transgenic animals. A second assay that will detect an endogenous mouse gene, such as beta-globin, or an endogenous rat gene, such as prolactin, is required in order to demonstrate that the DNA preparations are amenable to PCR. Animals are tested with both assays so that no transgenic founder is mistakenly discarded because the tail DNA is not suitable for PCR. A Southern blot assay is also needed. ^[7]

8. Establish an Expression Assay :

It's important to show that transgene is expressed. RNA expression can be detected by in situ hybridization or RNAse protection assay with RNA probes. Alternatively, an RT-PCR approach can be used.. ^[7]

9.Screen Potential Founders :

A genetic founder, denoted "F0," is a first-generation transgenic animal that develops directly from a microinjected embryo and carries the transgene stably integrated in its genome.

Transgene sequences are analyzed by biochemical and molecular methods to confirm that they are complete and intact. If they are, then the animals are raised to maturity and bred to non-transgenic mates. Their offspring are tested to confirm that the transgene is inherited at the expected frequency.

Milk is collected from these transgenic animals and analyzed to measure the expression levels and biochemical characteristics of the recombinant protein. Milk may be collected directly from female founders, or from the daughters of male founders.

Tail biopsies from potentially transgenic animals will be obtained 5 weeks after injecting eggs (3 weeks gestation time and 2 weeks of post-natal growth). DNA is extracted from the tail biopsies by simple salt out methods or you can use kits from various vendors on the market. For speed, some people use the "HotShot" method. Once the investigator has the DNA we expect you to test each DNA sample for both the transgene and an endogenous mouse gene or rat gene by PCR. Ideally, you will identify which pups are transgenic before they are weaned at three weeks of age so that only transgenic pups are moved to your animal room. If the testing is not complete then we will transfer all of the pups to your animal room. ^[7]

10 . Breeding and Analysis of Transgenic Rodents :

The final stage in the process is to study animals carrying the transgene. Typically, the transgenic founder animals are bred to mice of defined genetic background such as C57BL/6. Transgenic rats are bred to Sprague-Dawley since that is their originating genetic background. Analysis of transgene expression and the consequences of expression are generally conducted in the offspring. The best strategy, if applicable is transgenic founder analysis. This eliminates the time and cost of breeding multiple offspring from each founder. ^[7]

Examples Of Transgenic Animals:

The first transgenic animal, a mouse, was produced in 1981. In an effort to determine which genes were involved with cancer, a gene was inserted into the mouse that made it susceptible to cancer.

In 1985, the first transgenic farm mammal was produced, a sheep called "Tracy". Tracy had a human gene that expressed high levels of the human protein alpha-1-antitrypsin. The protein, when missing in humans, can lead to a rare form of emphysema.

Many more animal clones have been generated in the mean time. For example, cloned cows appeared in 1999 and now there are cloned pigs that have been modified to reduce transplant rejection of pig organs in humans. Cloned pets (cats and dogs) have been created too. There are even cloned mules.

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. ^[1]

GSK scientists engineered the overexpression of the human mitochondrial transporter protein, "uncoupling protein-3" (UCP-3), in skeletal muscle in mice. In this model, the transgenic mice were found to eat more than wild-type littermates, yet remain leaner and lighter. The mice also exhibit lower glucose and insulin levels and an increased glucose clearance rate, leading to the hypothesis that compounds that regulate expression of UCP-3 might be of use in treating obesity. ^[5]

In theory, large quantities of the human protein can be produced in the animal's milk and subsequently purified for use in medical therapies. It has been gaining application among biotechnologists since the development of transgenic "super mice" in 1982 and the development of the first mice to produce a human drug, tPA (tissue plasminogen activator to treat blood clots), in 1987.

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. ^[1]

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. ^[1]

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. ^[1]

Advantages:

The benefits of transgenic animals include:

· Large-scale, low-cost production independent of proximity to the oceans and with reduced environmental impacts;

·Increased growth rates;

·Improved disease resistance;

·Improved food-conversion rates;

·Leaner meat;

·Increased muscle mass;

·Improved wool quality;

·Improved nutritional quality or appeal; and

·More efficient use of indoor water-recycling plants.

Specifically, the environmental and nutritional benefits resulting from aquaculture outweigh potential risks, which include the runoff of pollutants into closed or semi-closed waterways; the elimination of coastal forests and ecosystems; the potential for increases in waterborne disease and parasites; and sustainability. ^[8]

Advantages Over Selective Breeding:

Transgenic technology is an extension of agricultural practices that have been used for centuries: selective breeding and special feeding or fertilizing programs. It may reduce or even replace the large-scale use of pesticides and long-lasting herbicides. Transgenic technology is still experimental and is still very expensive. However, it offers a number of advantages over traditional methods.

Compared with traditional methods, transgenic breeding is:

More specific – scientists can choose with greater accuracy the trait they want to establish. The number of additional unwanted traits can be kept to a minimum.

Faster – establishing the trait takes only one generation compared with the many generations often needed for traditional selective breeding, where much is left to chance.

More flexible – traits that would otherwise be unavailable in some animals or plants may be achievable using transgenic methods. Less costly – much of the cost and labour involved in administering feed supplements and chemical treatments to animals and crops could be avoided.

Environmentally friendly – allowing less use of chemical pesticides and herbicides and reduced tillage leading to less land degradation.

Overall, the use of transgenic technology has many advantages over traditional methods. Transgenic breeding is said to be more specific, faster, and less costly. Right now research is limited to traits involving one or a few genes. Before scientists can manipulate complex traits, there is going to be the need for many years of research.

Applications Of The Transgenic Animal

Research into transgenic animals could prove useful in several ways. Scientists can provide animals with beneficial genes or traits, such as disease resistance, that will improve their quality of life and bolster waning populations. Transgenic animals may also be designed for organ production, helping to ease the critical shortage of kidneys and livers available for transplants. In addition, scientists are researching ways to produce proteins or drugs in transgenic animals.

Applications of transgenic animals are described in detail in this section, which can be categorized in three groups:

Medicinal Applications:

1. Models of human disease processes:

One of the most important applications of transgenic animals is the development of new animal models for human disease. Gene targeting is being exploited by scientists to create models of human disease. The genetic setup of an animal may be modified in such a way that it develops a disease similar to an equivalent human disease.

Hundreds of transgenic rodent lines have been produced by introducing into the genome genetic sequences such as viral transactivating genes and activated oncogenes implicated in specific pathologies. Transgenic rodent models have been characterized for several human diseases including cardio-vascular disease (Walsh et al., 1990), cancer (Sinn et al., 1987), autoimmune disease (Hammer et al., 1990), AIDS (Vogel et al., 1988), sickle cell anemia (Ryan et al., 1990), muscular dystrophy, Lou Gehring’s disease, and neurological disease.

Here are some examples of transgenic animals developed as models of human disease:

Transgenic animals can serve as models for many malignant tumors. Inserting the c-myc oncogene, which regulates cell growth, into a mouse creates a transgenic strain with a high rate of spontaneous tumors. The type of tumor depends on the promoter placed in front of the c-myc gene in the contruct. The mammary tumor virus (MTV) promotor increases the incidence of breast adenocarcinomas. The immunoglobulin heavy-chain enhancer (IgH), when inserted along with the c-myc, results in a strain of mice with a high incidence of lymphoblastic lymphomas. Although mice have been the most frequent hosts for transgenic modification, other domestic animals have also been used.

Transgenic mice overexpressing the amyloid precursor protein form deposits in the brain that resemble the amyloid plaques found in Alzheimer's patients. Mouse models such as these can potentially be used to test drug therapies and to learn more about the progression of the disease.

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. ^[1]

Transgenic animals enable scientists to understand the role of genes in specific diseases. By either introducing or inactivating particular genes, researchers can often for the first time discover the root causes of diseases associated with gene defects. For example, GSK scientists engineered the overexpression of the human mitochondrial transporter protein, "uncoupling protein-3" (UCP-3), in skeletal muscle in mice. In this model, the transgenic mice were found to eat more than wild-type littermates, yet remain leaner and lighter. The mice also exhibit lower glucose and insulin levels and an increased glucose clearance rate, leading to the hypothesis that compounds that regulate expression of UCP-3 might be of use in treating obesity. ^[5]

Reasons for using the transgenic animal as a model for human diseases:

Transgenics may spare the use of higher animals. The creation of transgenic animals is resulting in a shift from the use of higher order species to lower order species. In the long term, a reduction in the number of animals used, for example to study human diseases is possible due to a greater specificity of the transgenic models developed. This shift in the patterns of animal use is being monitored by the CCAC through the use of the Animal Use Data Form. An example of the replacement of higher species by lower species is the possibility to develop disease models in mice rather than using dogs or non-human primates. ^[2] On the other hand, the success of the method has led to using its potential for investigating a wider range of diseases and conditions. The actual use of some species may be increased. ^[5]

2.As organ transplant donors to humans:

Another rapidly-moving field is the potential use of transgenic pigs for use as organ transplant donors to humans. 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, xenotransplantation 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. ^[1]This kind of research is still very much in its infancy. If successful, however, this research could transform the lives of the many patients awaiting organ transplants.

Transgenic animals are being developed by some companies to provide new organs for transplantation such as kidneys, livers and hearts. Transgenic pigs with human histo-compatibility genes have been bred in the hope that their "humanized" organs will not be rejected by a patient's immune system. ^[10]

3. Proteins of medical importance to humans:

One important application of transgenic technology is the generation of transgenic livestock as "bioreactors." Transgenic animals can produce biological products. It may be possible to use transgenic animals to make rare biological products for medical treatment. Milk-producing transgenic animals are especially useful for medicines. Key human genes have been introduced into sheep, cows, goats, and pigs so that the human protein is secreted into the milk of the transgenic animal. In theory, large quantities of the human protein can be produced in the animal's milk and subsequently purified for use in medical therapies. ^[15]

Principle behind the technique:

The major function of the mammary gland is to produce proteins. The mammary gland is capable of producing milk that carries over 40g/L of protein. Advantage of the unique properties of this "natural protein secretion organ" is taken in this technique. By utilizing molecular biology technology, we can design DNA constructs that reliably express high levels of therapeutic proteins in the milk of the animals that carry the transgene. Advantage of the normal mammalian protein processing mechanisms is taken to synthesize properly folded and assembled complex proteins. Although the epithelial cells in the mammary gland do not usually express antibodies, it has been found that the machinery needed to properly fold and assemble the heavy and light chains of antibodies are well represented in these cells. By utilizing the milk specific promoters to express the heavy and light chains, the cellular machinery is capable of secreting high levels of properly folded antibody.

Since this is a mammalian cell system, it is capable of post-translational modifications such as glycosylation and gamma carboxylation. Many recombinant proteins, most of which are of human origin, require glycosylation for proper function or pharmokinetics. This system provides high level expression combined with mammalian modifications-unique to production systems. This method permits flexible scale-up of protein manufacturing to meet increasing production needs throughout the product development process. Scale-up is as simple as breeding more transgenic animals. This is easier and less expensive than building and validating a larger biopharmaceutical fermentation or mammalian cell culture facility, therefore reducing overall capital costs.

Examples:

An early example of this technology by John Clark and colleagues was the production of transgenic sheep expressing the human blood-clotting factor IX needed by many patients with hemophilia. These researchers placed the human factor IX gene under the control of a piece of sheep DNA that normally turns on the beta-lactoglobulin gene in the mammary tissue. Though the sheep secreted factor IX into their milk, the levels of the protein were very small. With advances in the efficiency of creating and expressing genes in transgenic farm animals, therapeutic proteins can now be isolated. ^[15]

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. ^[1]

Human alpha-1-antitrypsin, a protein used to treat the rare genetic disorder of alpha-1-antitrypsin deficiency, is also produced by this technique. ^[1]

Other human proteins that have been expressed in transgenic animals include: anti-thrombin III (to treat intravascular coagulation), collagen (to treat burns and bone fractures), fibrinogen (used for burns and after surgery), human fertility hormones, human hemoglobin, human serum albumin (for surgery, trauma, and burns), lactoferrin (found in mother milk), tissue plasminogen activator, and particular monoclonal antibodies (including one that is effective against a particular colon cancer). Animals mostly used for this work are pigs, cows, sheep, and goats. ^[3]

Comparison with other methods of protein production:

There are four other means of commercial protein production. E. coli production, which was the first commercialized, is very efficient, but limited to simple non-glycosylated proteins. Although the cost of production is low, the cost of processing and refolding the proteins is significant.

Fungal systems, such as Pichia or filamentous fungi allow efficient production of some secreted proteins, but the glycosylation is usually high mannose which can affect the pharmokinetics of the protein.

There is also the baculovirus production system, which can produce a wide range of proteins in small scale, but has yet to be scaled up to commercial levels.

The standard method for producing complex glycosylated proteins, (i.e. Monoclonal Antibodies) is with cell tissue culture. The protein may be properly folded and modified, but the low yields per cost of production facility limit the number of proteins that can be developed.

Recombinant protein concentrations in the milk of transgenic animals are substantially higher than levels attained in cell tissue cultures. Expression levels of 2 to 10 grams of recombinant protein per liter of milk are readily achievable in transgenic livestock. In comparison, highly optimized cell cultures can typically generate 0.2 to 1 gram per liter of culture medium. It appears that transgenic technology can achieve the high levels of recombinant protein production normally found only in prokaryotic systems. It has the added benefit in that it is a mammalian system that can secrete complex, glycosylated proteins, similar to tissue culture. Thus it has the best of both technologies, with the added advantage of lower capital cost for the production facility.

Transgenic production takes advantage of normal mammalian protein processing mechanisms to synthesize properly folded and assembled complex proteins -- all within the cells of the mammary gland. This method permits flexible scale-up of protein manufacturing to meet increasing production needs throughout the product development process. Scale-up is as simple as breeding more transgenic animals. This is easier and less expensive than building and validating a larger biopharmaceutical fermentation or mammalian cell culture facility, therefore reducing overall capital costs.

Recombinant protein concentrations in the milk of transgenic animals are substantially higher than levels attained in cultures of yeast, bacteria, insect cells or mammalian cells. Expression levels of 2 to 10 grams of recombinant protein per liter of milk are readily achievable in transgenic livestock. In comparison, highly optimized cell cultures can typically generate 0.2 to 1 gram per liter of culture medium.

Animal most commonly used for the purpose of protein production:

In choosing a species of animal it is optimal to have animals that have been bred for significant milk production and also have a relative short generation time. Formally, choices range from mice, one of the model systems with a generation time of 3 months and milk yield of 1 ml, to rabbits with an 8 month generation time and 4 liter yield, to the largest commercial species, the cow. Cows have a generation time of 3 years, with an annual milk yield of 8000 liters.

Since time is critical, goats are a logical alternative with a generation time of 18 months and a yield of nearly 800 liters. As a dairy breed, goats show efficiency of milk production that is unrivaled. As a dairy production animal goats are utilized all over the world. Significant expression, (2-10g/L) of recombinant proteins in lactating goats has been shown. With an annual yield of 800 liters, over 1 kilogram of recombinant protein can be produced per lactating animal. The scale-up of the goat herd following standard breeding is straight-forward and the production of 100's of kilograms of recombinant proteins can be readily achieved. This is well within the levels expected for most recombinant protein markets. Their dairy characteristics combined with their relative short generation time allow meeting our goals. Small amounts of the recombinant protein for initial testing can be delivered within a year, followed by high levels during normal lactation. By utilizing a known dairy animal, the scale-up for large volume production is straightforward. Goats are an ideal dairy species as produce large volumes of milk with high protein content, and are generally accepted as a source of dietary milk. They are relatively easy to breed and maintain. Goat milk has been extensively characterized biochemically, and this makes it more straightforward to develop protein purification procedures.

4. To test the safety of new medicines and vaccines:

Because transgenic models can highlight specific characteristics such as certain mechanisms involved in the formation of tumors, they can demonstrate more clearly the possible side effects of new therapies. Their use in early toxicity trials may also serve to prevent the subsequent use of a larger number of animals in the development phase. Toxicity-sensitive transgenic animals have been produced for chemical safety testing.

Transgenic animals can also be used to test the identity and purity of human proteins used as drugs. A transgenic animal that makes a human protein (e g human insulin) will recognise this substance as its own and will therefore not produce an immune response against it. As a consequence, the identity and purity of the product can be tested more efficiently in such animals, thereby saving the use of many laboratory animals otherwise needed to obtain a statistically significant result. ^[13]

5. Genatic Research

Genetic models to study the effects of genetic changes on development:

Frequently used in genetic research are transgenic fruit flies (Drosophila melanogaster) as genetic models to study the effects of genetic changes on development. Flies are often preferred over other animals for ease of culture, and also because the fly genome is somewhat simpler than that of vertebrates. Transgenic mice are often used to study cellular and tissue specific responses to the disease. ^[15]

Agricultural Applications:

6. 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. ^[1]

7. Production of milk low in cholesterol:

Transgenic cows exist that produce more milk or milk with less lactose or cholesterol. ^[1]

8. The polled (hornless) condition in cattle. ^[10]

Industrial Applications:

9. material fabrication:

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. ^[1]

10.Increased Meat Production:

An abnormally high quantity of growth hormone in the transgenic animal is responsible for increased meat production. Pigs and cattle that have more meat on them can be produced. 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. ^[1]

11. Sheep that grow more wool:

Breeding transgenic sheep that grow better wool without needing dietary supplements of sulphur-containing amino acids is under research.

The Future:

Research is presently limited to traits involving one or a few genes. It will probably require many years of research before scientists can manipulate complex traits (such as meat quality or animal behaviour) that are influenced by many genes.

Much current research focuses on the understanding and developing useful promoter sequences to control transgenes and establishing more precise ways to insert and place the transgene in the recipient. Much still needs to be done to improve our knowledge of specific genes and their actions and of the potential side effects of adding foreign DNA and of manipulating genes within an organism.

Reference:

1. www.actionbioscience.org/biotech/margawati.html

2. www.acs.ucalgary.ca/~/browder/transgenic.html

3. http:/photoscience.la.asu.edu/photosyn/courses/BIO_343/lecture/transan.html

4. www.accessexcellence.org/AB/BA/casestudy3.html

5. www.gsk.com/research/about/about_animal_roles.html

6. www.bookrags.com/research/transgenic_animal_gen_04

7. www.med.umich.edu/tamc/tgoutline.html

8. http://bio.org/animals/faq.asp

9. www.cartage.org.lb/en/themes/Sciences/Zoology/Animal Pathology/TransgenicAnimals.html

10. www.biotopic.co.uk/edexcel/biotechnology/trans.html

11. www.ehs.uiuc.edu/bss/factsheets/transani.aspx?+bID=fs

12. www.accessexcellence.org/RC/AB/BA/Casestudy4.html

13. www.novonordisk.com/sustainability/positions/transgenic_animals.asp

14. www.transgenics.com/products/questions.html

15. www.biotech.iastate.edu/biotech_info_series/bio10.html

About Authors:

M s . Neha Vithani is Post Graduate student of IIT, Kanpur . She has done her graduation from Institute of Pharmacy , Dept. of Pharmaceutics & Pharm. Technology, Nirma University of Science & Technology, Ahmedabad, Gujarat . She occupied 7^th Rank in GATE examination in all over India . Her research interest includes Biotechnology, Genetic engineering.

Mr. Jigar N. Shah is currently working as Lecturer at Nirma Institute of Pharmacy, Dept. of Pharmaceutics & Pharm. Technology, Nirma
University , Ahmedabad, Gujarat . His current job responsibilities include teaching UG, PG classes as well as doing research for Ph. D. He presented his research work in a number of National & International conferences. He has 2 publications in International Journal. His research interests include Ocular Drug Delivery Systems, Nanotechnology and Transdermal Drug Delivery Systems. He is a Life Member of Association of Pharmaceutical Teachers of India.