The advancement in bioscience to produce transgenic animals by introducing the foreign DNA into the germ line of an animal has elevated the potential of biological research to an unprecedented level. There are four principal methods used in the pursuit of producing transgenic animals and they are DNA microinjection, embryonic stem cell mediated gene transfer, retrovirus mediated gene transfer and nuclear transfer. The foreign DNA can be inserted physically into the nucleus of host cells with very small syringe or with minute particles fired from a gene gun. The success rate in terms of live birth of animals containing the transgene is extremely low. However, the Testis mediated gene transfer (TMGT) has resulted a success rate of >94% in mice which is very high. Transgenic animals are the crux of various pursuits that benefit human. These are mainly utilized as transpharmers, xenotransplanters, scientific models, diseases models and food sources.
Numerous bio-techniques conceived in the recent past have revolutionized the animal production scenario and transgenic animal production is one among them. Basically, transgenic animal production, involves the genetic modification i.e. insertion or deletion of one or more genes. Horizontal gene transfer i.e. insertion of genetic material from different species, can naturally occur. The prerequisites to mimic this artificially are the vectors (e.g. viruses, plasmids) that carry the attached genes of our interest. Otherwise, the foreign DNA can be inserted physically in to the nucleus of host cells with very small syringe or with minute particles fired from a gene gun. Paradoxically, other methods exploit natural forms of gene transfer, such as the ability of Agrobacterium to transfer genetic material to plants, or the ability of certain viruses viz. lentiviruses, adenovirus, retroviruses, to transfer the genes to animal cells. Transgenic animals are known commonly as “transgenics”, alternatively also known as “Genetically Modified Organism” (GMO) or “Genetically Engineered Organism” (GEO). These are just one in a series of developments transpired in the area of animal biotechnology, whose genetic material has been altered in a premeditated manner using genetic engineering techniques. 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 (FELASA, 1995). Transgenic animals, in other words, are the one that carries a foreign gene that has been intentionally inserted into its genome.
Transgenic Animal Production Methods
There are four principal methods used in the pursuit of producing transgenic animals and they are DNA Microinjection (Gordon et al., 1980), Embryonic Stem Cell mediated Gene Transfer (Torres, 1998), Retrovirus mediated Gene Transfer (Jaenisch, 1976) and Nuclear Transfer (Wilmut et al., 1997). Each of these techniques has been successful in generating small animals and in particular, transgenic mice. Attempts to introduce transgenes into larger animals, mainly livestock by these same methods had limited success (Chang et al., 2002; Houdebine, 2002) due to longer gestation period and generation times, reduced litter sizes and the large numbers of donor animal necessary (Mullins and Mullins, 1996). These methods are technically difficult, require costly equipment and highly specialized training. The most widely used method is DNA microinjection (Gordon et al., 1980). However, this method has a very limited success rate (<1%) in large animals. It does also require great technical skill and intensive labour (Sato, 2005). The success rates of embryonic stem cell mediated gene transfer and nuclear transfer methods are only 2% (Gossler et al., 1986) and 1-3% (Solter, 2000), respectively. In the following sections, of transgenic animal’s production and their possible utilities in enhancing the human health status are briefly deliberated.
Microinjection of DNA
This is the most popular way to make transgenic animals and is one of the first and an effective method in mammals (Gordon and Ruddle, 1981). In this method, single gene or a combination of genes from other members of the same species or from a different species will be constructed and is injected directly onto the male pro-nucleus of a fertilized ovum by holding the egg stable by microtube suction device while a solution containing 200-300 copies of the transgene is injected using a micropipette. Injection of transgene into the male pronucleus is due to its larger size than the female pro-nucleus. This manipulated fertilized ovum is transferred into the oviduct of a foster mother that has been synchronized to act as a recipient by mating with a vasectomized male. 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.
Material and Method
Construction of Gene of Interest
The exotic gene is constructed using recombinant DNA methodology. In addition to structural gene, vector DNA sequences that enable the incorporation of imported molecules into host genome, promoter and enhancer sequences that facilitate the proper expression of imported gene in the host cells will be included in the construct.
Transformation of Fertilized Eggs
After harvesting the freshly fertilized eggs, inject the prepared DNA construct into the male pro-nucleus prior to the sperm head becoming a pro-nucleus. When the male and female pro-nuclei have fused to form the diploid zygote nucleus, allow the zygote to proliferate to form a two-cell embryo and then select the successfully transformed cells.
Implantation of Embryos
Stimulus of mating with vasectomized males elicits the hormonal changes that are imperative to make the recipient uterus receptive. Then transfer the manipulated embryos into pseudo-pregnant foster females and anticipate the successful implantation of embryos. Out of the transferred embryos, one third develops into healthy offspring.
Test the Offspring
After the birth of offspring, it is probed for the presence of the extraneous DNA by collecting a small piece of tissue, may be from tail, using molecular biology tools such as Polymerase Chain Reaction (PCR), Reverse Transcription- Polymerase Chain Reaction (RT-PCR) or Southern blot analysis. No more than 10 to 20 % of the born offspring will have the introduced gene and they will be heterozygous for that gene.
Establishment of a Transgenic Strain
The progenies born by mating the two heterozygous individuals will be screened for transgene and one offspring out of four will be homozygous. Mating these homozygous individuals again will form the transgenic strain (Fig. 1).
Fig.1: DNA microinjection method of producing transgenic animals
Embryonic Stem Cell-Mediated Gene Transfer
This is the second method of producing transgenic animals and is a choice to insert a transgene to a specific site in geneome. It involves prior insertion of desired DNA sequences via microinjection, a virus, certain chemicals, or homologous recombination into an in-vitro culture of embryonic stem (ES) cells. These ES cells, harvested from the inner cell mass (ICM) of blastocysts, prepared by in vitro fertilization or collected from female, can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes. Once transformed, ES cells can be left in-vitro to split or injected into developing embryo at blastocyst stage and implanted into a host’s uterus get chimeric animal. Further, chimeras should be crossed to get the transgenic individuals. This technique is of particular importance in the study of the genetic control of developmental processes and has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination and works well particularly in mice. Also, this technique is the method of choice for gene inactivation, the so-called knock-out method.
Virus-Mediated Gene Transfer
Gene transfer that is mediated by a carrier or vector, generally a virus or a plasmid increases the probability of gene expression. Among the many viruses, retroviruses are used commonly to transfer genetic material into the cell by taking the advantage of its ability to infect host cells. Transgene transmission is possible only if the retrovirus integrates into some of the germ cells. Offspring derived from this method are chimeric, i.e. not all cells carry the retrovirus.
Transfer of nucleus form a totipotent donor cell into enucleated mature oocyte and resulting transfer of embryo to surrogate mother for development to term. Nuclear transfer using embryonic, fetal and/or somatic cells as karyoplasts resulted in the production of cloned animals.
Sperm Mediated Gene Transfer
In the process of inventing new and efficient methods of transgenesis, researchers have developed new method known as Sperm mediated gene transfer (SMGT) in which sperms are used as vector. In this method, sperm cells are exposed to foreign DNA, which binds to the surface of the sperm through specific protein-protein interactions. The DNA associated with the sperm is then spontaneously incorporated via protein interactions into the sperm nuclei (Zani et al., 1995) and is incorporates it into egg in a process of fertilization (Celebi, 2003). Spermatozoal competence to take up and transfer the exogenous DNA was proved first time in rabbit (Bracket et al., 1971). SMGT, over the time, including its alternative approach testis mediated gene transfer (TMGT), has become an established and reliable method for transgenesis. These methods have been operated extensively to produce transgenic animals and offers exciting prospects for experimental and applied biology, agricultural and medical sciences. Successful SMGT has been reported in various species viz. crustacean (Chen et al., 2006), fishes (Khoo, 2000; Kurita et al., 2004), amphibians (Jonak, 2000), aves (Gruenbaum et al., 1991; Nakanishi and Iritani, 1993), and mammals (Lavitrano et al., 1989; Seperandio et al., 1996). In the year 2006, the bull spermatozoa have been successfully transfected by Anzar and Buhr (2006). They have reported the greater efficiency of transfection in frozen-thawed semen than fresh spermatozoa. The same results have also been reported in goat (Zhao et al., 2005). Numerous researches have been underpinned to increase the sperm capability to capture the foreign DNA by chemical as well as physical methods. Chemical methods mediated transgenesis includes utilization of liposome and linkers which have low success rate such as 0% for liposome mediated (Bachiller et al., 1991) and 20% for linker based methods (Epperly, 2007). Liposomes have been used in mouse (Bachiller et al., 1991), rooster (Rottmann et al., 1992), rabbit (Sperandio et al., 1996), bull (Sperandio et al., 1996) and chicken (Sperandio et al., 1996). In contrast, physical method primarily includes electroporation (Gagne et al., 1991) that is used mainly to investigate the spermatozoal ability to carry the exogenous DNA into the bovine egg rather than the production of genetically modified animals. Electroporation was used in bull (Gange et al., 1991), pig (Horan et al., 1992), and salmon (Sine et al., 1993; Symonds et al., 1994). This Electroporation is a method known as Testis mediated gene transfer (TMGT) has resulted in a success rate of >94% in mice when used for in-vivo transfection of sperms (Dhup and Majumdar 2008).
Since its inception, SMGT has been a highly controversial issue (Lavitrano et al., 1989), because several groups have reported the failure to reproduce the original protocol (Brinster et al., 1989). Currently, only two steps in the SMGT process are well-established and fully reproducible: (i) the spontaneous interaction between sperm cells and foreign DNA molecules, and (ii) the delivery of sperm-bound DNA to oocyte at fertilization. The subsequent fate of sperm-bound DNA, after delivery in the oocyte, is still a contradictory issue; in particular, the question of whether foreign molecules of nucleic acids become integrated into the host genome or remain as extra chromosomal structures is still unsolved. Therefore the performance of SMGT, if improved could be one of the most rapid ways to produce transgenic animals. For any of these techniques the success rate in terms of live birth of animals containing the transgene is extremely low. Providing that the genetic manipulation does not lead to abortion, the result is a first generation (F1) of animals that need to be tested for the expression of the transgene. Depending on the technique used, the F1 generation may result in chimeras. When the transgene has integrated into the germ cells, the so-called germ line chimeras are then inbred for 10 to 20 generations until homozygous transgenic animals are obtained and the transgene is present in every cell.
Detection of Transgene in Progenies
A variety of methods have been employed to determine the presence of foreign DNA in the developing animal. PCR or Southern blotting of genomic DNA can measure the presence of foreign DNA in the blastocyst, embryo or young animal (Lavitrano et al., 1989; Rottmann et al., 1992). The expression of the transgene is usually estimated by measuring the enzyme activity coded by the transgene (chloramphenicol acetyl transferase, -galactosidase, green fluorescent protein etc) (Lavitrano et al., 1989) or by Western analysis or enzyme-linked immunosorbent assay (ELISA) of the gene products (Lavitrano et al., 2003).
Transgenic Animals Utility for Enhancement of Human Health
Transgenic animals are the crux of various pursuits that benefit human. They are mainly utilized as transpharmers, xenotransplanters, scientific models, diseases models and food sources.
Transgenic Animals as Organ Donors
Organs transplantation is done to retrieve the function of diseased organs and in that pursuit, histocompatibility of donor organs with the recipients determines the success of organ transplantation. However, only small percentage of donated organs is histocompatible with the recipients. Therefore, there is an intense paucity of matched organs. This scarcity is overcome by administering immunosuppressive drugs to patients/recipients which jeopardize the patients to infections. With loads of shortcomings in the conventional organ transplantation, xenotransplanters have significant role in conquering the organ shortage. These xenotransplanters are animals that have been engineered genetically to not to express the foreign antigen that normally prevents the transplantation of their organs into humans, there by provide the animal organs that are histocompatible with humans. Among the animals, pig was chosen xenotransplant research with the rationale of its similar physiology with humans. Even though xenotransplanter pig has been produced already, human trials have not been approved yet.
Transgenic Animals as Disease Models
With greater specificity of the transgenic models developed, study of human diseases using modified animals has become reality. Disease models are animals engineered to express the symptoms, progression of a particular disease so that treatments for that disease can be tested on them. Some of the disease models that have been developed includes AIDS mouse, Alzheimer’s mouse, oncomouse (a model for cancer), and Parkinson’s fly. These genetically modified animals have provided the tools for exploring many biological questions of diseases affecting humans. An example; normal mice cannot be infected with polio virus as they lack the cell-surface molecule that, in humans, serves as the receptor for virus. Therefore, normal mice cannot serve as an inexpensive, easily-manipulated model for studying this 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. Transgenic animals, therefore, can be used, instead of humans, as a model to study the intricate molecular mechanism of various incurable diseases affecting humans, which eventually are invaluable in designing or formulating the drugs, developing vaccines and to explore other proteins of pharmacological importance. Also, transgenic mice are often used to study cellular and tissue-specific responses to disease.
Transgenics as Food Source
Animals farming for food would be propitious if they could grow more efficiently with fewer resources and in less time. With long time required in conventional production of animals that grows bigger and faster with greater efficiency, transgenic animals will be an alternative strategy to prevail over the malnutrition and under-nutrition affecting humans hitherto. Some of the transgenic animals produced as food source are super-pig, which was a failure due to the large list of health issues, and super-fish, which is more promising.
Most of the transgenic superpigs were made by microinjection of the transgene for a growth hormone, whether porcine, ovine, bovine, or even rat. The famous Beltsville pig was made in Beltsville, Maryland under the supervision of the US Department of Agriculture. These pigs expressed human or bovine growth hormone and expressed higher levels of growth factors. Unfortunately, the Beltsville pigs had many health problems, the most commonly quoted one being arthritis. Animal rights groups claim that the pig also was impotent and had ulcers, heart problems, lameness, kidney disease, and pneumonia. The pigs were euthanized, and biologists imposed a voluntary moratorium on performing any further studies on mammals involving growth hormone.
Superfish and Supersalmon
Another attempt at a more efficient food source was a fish. One species of the fish, the Tilapia, was engineered by microinjection at Cuba to over express its own growth hormone. This animal was not transgenic, but it was genetically engineered. It showed accelerated growth, but it reached an adult size no larger than normal tilapia. Similar techniques have been used on salmon. The transgenic salmon produce the growth hormone continuously, instead of turning it off depending on the season. The eggs of a species of usually slow-growing trout were microinjected with the gene of a salmon that grew quickly after many generations of selective breeding. Because of a concern over the escape of these fish into the environment, a very tight control is kept over transgenic fish farms. The biggest fear is that these fish will breed out native fish because they would be able to out match them for food. There is considerable opposition to the creation and farming of these “superfish”. But the transgenic fish look like a much more likely source of food than any transgenic animal species.
Transgenic Animals as Transpharmers
In discovering and development of cures and therapies for many diseases affecting humans, transgenic are becoming vital. By modifying or transferring the DNA to dairy animals, a protein(s) are expressed or over-expressed in the mammary gland so that their milk contains the required protein. First, this was done in blood; however, the direction has switched to milk due to the relative ease with which the drugs acquired from milk and the proteins expressed in milk are less likely to affect the human physiology. Prior to pursue with large animals like cows and goats, due to the difficulty in performing in-vitro fertilization and foster motherhood, transpharming transgene will be tested first in mice to ensure the encoded functional proteins.
Some examples of transpharming are- In mouse, the first transpharmer, clot dissolver drug tissue plasminogen activator (tPA) was expressed and human-alpha-antitrypsin, a inhibitor to treat emphysema, was produced in mouse milk. In rats, a deliberate modification to secrete human-alpha-lactalbumin in their milk has yielded the result as anticipated. In sheep, Human-alpha-1-antitrypsin has been produced and used as therapeutic agent in humans with deficiency and human clotting factor was secreted in the milk of ewes. The first transpharmer goats were created in 1991 to produce tissue plasminogen activator, a clot dissolving drug. In 1999, using somatic cell nuclear transfer (SCNT) the then new process, transpharmer goats were produced that not only over-expressed the intended gene encoding human antithrombin III, a kind of anticoagulant, but also passed down that transgene to their offspring. In the milk of cloned transgenic cattle Bioactive Recombinant Human Lysozyme was expressed. Numerous medicinal agents including insulin and many immunizations have been developed in transgenic animals. Transgenic sheep and goats, and chickens are now capable to manufacturing the exogenous proteins in their milk and white, respectively, which are of some use to humans.
To Study the Intricate Mechanisms of Biology
Scientific or biological models are animals with some transgene deliberately introduced to their genome to study the genetics and expression of specific gene(s) or some natural process. Transgenics allows us to ascertain the purpose of proteins in biological mechanisms or development, which can in turn be applied to humans. Also, transgenic animals provide a means of evaluating genetic modifications in terms of anatomical and physiological changes in a complex system because of the non-reproducible complex interactive processes of the living beings in-vitro. Transgenic models are more precise in comparison to traditional animal models, for example the oncomouse that is susceptible highly to tumor development enables us to procure the results for carcinogenicity studies within a shorter time-frame, which eventually reduces the course of tumor development in experimentally affected animals. However, these models are not comparable absolutely; therefore in delineating conclusions from these data care must be taken. Transgenic mice have provided the tools for exploring many biological questions, especially genetic control of development. In medical research, transgenic animals are used to identify the functions of specific factors in complex homeostatic systems through over- or under-expression of a modified gene (the inserted transgene). In molecular biology, analysis of the regulation of gene expression makes use of evaluation of a specific genetic change at the level of the whole animal.
Transgenesis is very expensive still it is growing rapidly and new pharming uses are being discovered and developed. The success of this technology has led to using its potential for investigating a wider range of disease conditions that are inflicting humans and to manufacture proteins of great importance in ascertaining cures and treatments. Therefore, there is a pressing need to refine the transgenic techniques exquisitely so as to enhance the success rate and to reduce the cost of modified animal production. If this is materialized, the merit of this technology is inestimable.