INTRODUCTION

Also, Yeast Artificial Chromosomes YACs transgenic mice are generated by using pronuclear microinjection and represents latest generation of vectors which have the great advantage of large insert size. This method succeeded in mice and rabbits. D Only a spermatogonial stem cell can generate a colony of spermatogenesis in the recipient testis.

When testis cells carry a reporter transgene that allows the cells to be stained blue, colonies of donor cell-derived spermatogenesis are identified easily in recipient testes as blue stretches of tubule. Lentiviral Transfer of Oocytes And Zygotes: This method is used to overcome previous limitations of viral mediated gene transfer, containing the silencing of the transgenic locus and low expression levels Example including, generation of transgenic cattle by lentiviruses requires microinjection into the oocytes Recently H.

Sang from Roslin Institute has reported a different approach to overcome the problem associated with retroviral vectors. This study employed lentiviral based vectors. The technique was successful in showing about fold increases in the level of transgenesis Chimera Generation by injecting the Pluripotent Cells: Embryonic stem cells with pluripotent cells have ability to participate in organ and germ cell production after injection into the blastocysts Embryonic stem cells are important one for generating the gene knockins, large chromosomal rearrangements as well as gene knockouts As like embryonic stem cell, the another type of cells such as primordial germ cells are used for production of no.

As disease model: Historically, mice have been used to model human disease because of their physiological, anatomical and genomic similarities to humans. Transgenic animals are produced as disease models animals genetically manipulated to exhibit disease symptoms so that effective treatment can be studied such as Alzheimers, cancer, AIDS. Transgenic animals enable scientists to understand the role of genes in specific diseases.

The benefits of using transgenic animals include the possibility of the replacement of higher species by lower species- through development of disease models in mice rather than in dogs or non-human primates, the extent of discomfort experienced by parent animals during the experimental procedures. Transgenic animals such as mice have been found to be valuable in investifations into gene function and for analysis of different hereditary diseases 15, 38, As food: The FDA suggested that cloned animals and their products were safe to eat for human being Some drawbacks are associated due to their muscle hypertrophy like difficulties in calving requiring Caesareans, poor viability of calves and poor fertility.

Drug and Industrial production: Transgenic animals are used for production of proteins such as alphaantitrypsin, produced in liver, used in treatment of emphysema or cystic fibrosis. This process is less expensive than production of protein through culture of human cells The human lungs are constantly get affected by foreign particles such as dust, spores and bacteria.

To prevent these, neutrophils releasing the elastase enzyme but this enzyme harmed the elastin in the lungs which maintains the elasticity of lungs. Recombinant human proteins produced in the mammary glands of transgenic animals 43, Pharmaceutical proteins are now used for commercial purpose 45, Two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. The goats are used to manufacture silk, milk and secrete tiny strands from their body by the bucketful. By extracting polymer strands from the milk and weaving them into thread which is light and tough material that could be used to prepare military uniforms, medical micro sutures and tennis racket strings.

The mammary gland of transgenic goats is used to produce Monoclonal Antibodies. A recombinant bispecific antibody is produced by using transgenic cattles with in their blood Another application includes newly generation of trans-chromosomal animals in which a human artificial chromosome containing the complete sequences of the human immunoglobulin heavy and light chain loci was introduced into bovine fibroblasts, which were then used in nuclear transfer.

Transchromosomal bovine offspring were obtained that expressed human immunoglobulin in their blood. This could be a significant step forward in the generation of human therapeutic polyclonal antibodies Disease control: Scientist developed the mice by altering the genes of the mousepox virus in Australia Some scientist also thought to develop genetically modify mosquitoes so they cannot produce malaria but other scientist worry about these mosquitoes that they could have unforeseen possibly risk if, they are released into the environment Xenotransplantation: Now a day approximately about people are alive due to the successful transplantation of an appropriate allotransplantation.

Sometimes there is limitation of appropriate organs or rejection of live organ donation. So, to rectify this problem porcine xenografts from domesticated pigs are considered to be the best choice 52, Pigs which are genetically modified can be used as a source animal for tissues and organs in human beings for transplantation purpose by delete the gene responsible for the human rapid immune rejection response Survival rates, after the transplantation of porcine hearts or kidneys expressing transgenic regulators of complement activity proteins to immunosuppressed nonhuman primates, reached near about 23 to days.

So, the Hyperacute rejection can be overcome in a clinically acceptable manner For long term graft tolerance induction of permanent chimerism via intraportal injection of embryonic stem ES cells 58 or the co-transplantation of vascularised thymic tissue Blood replacement Transgenic swine are used to produce human haemoglobin. The protein obtained from transgenesis could be purified by using procine blood which is similar to human haemoglobin Transgenic pigs are used to produce pork by using spinach desaturase gene which produce large amount of non-saturated fatty acids, used for diet purpose and was advantageous to reduce the risk of stroke and coronary disease 63, Transgenic animals are used for milk production.

Generally, there is an improvement in milk composition. For this purpose transgenic mice have been developed, at the same time some unwanted side effects can occur 65, Transgenic pigs are use to increase milk production by altering the composition of lactose In the pig, transgenic expression of a bovine lactalbumin construct in sow milk has been resulting in higher lactose contents and greater milk yields, correlated with improved survival and development of piglets Transgenic sheep are used for wool production in which transgenic sheep carrying a keratin-IGF-I construct showed that expression in the skin and the amount of clear fleece was about 6.

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 The recent advances in reproductive technologies in vitro production of embryos, sperm sexing, somatic nuclear transfer, Lentiviral transfer of oocytes and zygotes, Chimera generation by injecting the pluripotent cells adds a new dimension to animal breeding. New and exciting techniques being developed will continue to expand this important and useful area of experimentation.

Use of Transgenic Animals in Biotechnology: Prospects and Problems

Download: Cited By: 0. Published: 01 September, Insertion of foreign gene may upset the expression of the genome. A desired characteristic of offspring could be established in one generation. Normal reproduction may result in a transgene being released to the wild population. The characteristic required can be chosen with greater specificity and accuracy. Embryonic Stem Cell-Mediated Gene Transfer 21, 22, 23 : In , the term embryonic stem cells ES cells were used to denote a cell line isolated directly from mouse embryos while, the term embryonal carcinoma cells EC were derived from teratocarcinomas.

Definition

They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes as shown in figure 1. Select for successfully transformed cells. Inject these cells into the inner cell mass ICM of mouse blastocysts.

Embryo transfer. Prepare a pseudopregnant The stimulus of mating elicits the hormonal changes needed to make her uterus receptive. Transfer the embryos into her uterus. Hope that they implant successfully and develop into healthy pups no more than one-third will. Test her offspring. Remove a small piece of tissue from the tail and examine its DNA for the desired gene. Establish a transgenic strain Mate two heterozygous mice and screen their offspring for the that will be homozygous for the transgene. Mating these will found the transgenic strain.

Retrovirus mediated gene transfer: Transgenic mice produced by retroviral transduction of male germ line stem cells. Male germ line stem cells have ability to self-renew and genetic modification of these cells would help to study the biology of their complex self-renewal and differentiation processes and to generate wide range of transgenic animal species Retroviruses used as vectors to transfer genetic material into the host cell, resulting into a generation of 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 when a simian virus was inserted into mice embryos, resulting in mice carrying this DNA Nuclear Transfer Method: In this method, the transgenic goats were produced by nuclear transfer of fetal somatic cells.

Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. Oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated.

In the second procedure, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Transfection of Gametes: The first transfection procedures occurred in the early s and experiments with different cell types and tissues has now become widespread.

Different transfection methods have been employed: a The in vitro procedure when foreign genes are introduced into cultured cells or tissues. Artificial Chromosome Mediated Gene Transfer 29 : A group of nuclei injected with transgene DNA, the eggs are transferred in medium of incubation and visual evaluation within next few hours. An individual animal develops after receiving the transgene DNA is referred as founder of a new transgenic lineage.

Transgenic animals

Testis cell transplantation method 30 : Testis cell transplantation method is shown in figure 2 and its steps are as follows: A A single-cell suspension is produced from a fertile donor testis. B The cells can be cultured C Microinjected into the lumen of seminiferous tubules of an infertile recipient mouse. E Mating the recipient male to a wild-type female F Produces progeny, which carry donor genes. Applications of Transgenic Animals: As disease model: Historically, mice have been used to model human disease because of their physiological, anatomical and genomic similarities to humans.

Transgenic animals are used in toxicity testing. Transgenic animals are used for vaccine testing. E, Pursel V. G, et al: Production of transgenic rabbits, sheep and pigs by microinjection. Nature ; Jaenisch R: Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc Natl Acad Sci. Gordon J. W, Scangos G. A, Plotkin D. J, Barbosa J. A, Ruddle F. H: Genetic transformation of mouse embryos by microinjection of purified DNA. Hammer R. M, Dolci S, Farace M. Cell ; 57 5 Chang K, Qian J, et al: Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

BMC Biotechnol. Perry A. Science ; Clark J, Whitelaw B: A future for transgenic livestock. Bowen R. A: Efficient production of transgenic cattle by retroviral infection of early embryos. R, BonDurant R. H, Behboodi E, Anderson G. B: Isolation of pluripotent stem cells from cultured porcine primordial germ cells. Cambridge University Press. Cambridge, UK, Gordon, J. W: Transgenic technology and its impact on laboratory animal science. Scandinavian Journal of Laboratory Animal Science ; Ben Mepham, Robert D.

Environ Mol Mutagen ; — Vijg J. Douglas GR: Bacteriophage lambda and plasmid lacZ transgenic mice for studying mutations in vivo. In: Pfeifer GP ed. Technologies for detection of DNA damage and mutations. New York, Plenum Press, ; — Ebert, K. M, and Schindler J. S: Transgenic farm animals: Progress report. Theriogenology ; — Gossler et al: Transgenesis by means of blastocyst-derived embryonic stem cell line, Proceedings of National Academic Science; Brinster, et al: Transgenic mice produced by retroviral transduction of male germ-line stem cells.

PNAS ; 98 23 — Proceedings of National Academic Science ; Alexander Baguisi et al: Production of goats by somatic cell nuclear transfer. Nature Biotechnology ; Esponda P: Transfection of gametes. 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.

Importance

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. 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. 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.

Biotechnology - applications part 3 - transgenic animals, Ethical issues

Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells. 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. At this stage embryos carrying the transgene can be frozen and stored for subsequent implantation.

Transgenic animals are just one in a series of developments in the area of biotechnology. Biotechnology has transformed the way in which we understand processes such as engineering and manufacturing. These terms now include the use of living organisms or their parts to make or modify products, to change the characteristics of plants or animals, or to develop micro-organisms for specific uses. The novel uses of biological techniques such as recombinant DNA techniques, cell fusion techniques, mono and polyclonal antibody technology and biological processes for commercial production have altered traditional distinctions and methods US Congress, Office of Technology Assessment, Genetic manipulations at the level of DNA have also changed long held views as to what is considered to be animal, plant and human.

In turn, these changes have made it more difficult to evaluate the ways in which animals are used and have obscured distinctions between pure and applied research. Consideration of the acceptability of creating specific transgenic animal strains or genetic manipulation involving interchanging DNA between species and kingdoms could be a simple animal care issue or a societal decision. The following is an attempt to show what the ability to create transgenic animals or engage in other forms of DNA manipulation means in terms of traditional ACC functions, not forgetting that this impacts on wider considerations of human responsibility for the welfare of other life forms.

The creation of transgenic animals is resulting in a shift from the use of higher order species to lower order species, and is also affecting the numbers of animals used. 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. 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.

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, in addition to the numbers of animals which are sacrificed as donors during the creation process. The potential of the technology has also made it possible to consider employing cattle, swine, sheep and goats as processing units to manufacture proteins or as organ donors. The complex interactive processes of living mammals are not reproducible in vitro.

However, transgenic animals provide a means of evaluating genetic modifications in terms of anatomical and physiological changes in a complex system.

References and Recommended Reading

Transgenic models are more precise in comparison to traditional animal models, for example the oncomouse with its increased susceptibility to tumor development enables results for carcinogenicity studies to be obtained within a shorter time-frame, thus reducing the course of tumor development in experimentally affected animals. However, models are not strict equivalents, so as with any other system care must be taken in drawing conclusions from the data.

A representative, but non-inclusive, list of purposes for which transgenic animals have been used indicates the wide ranging application of this biotechnology:. Important general considerations include the extent to which experience acquired in the laboratory with regard to husbandry should influence industry standards for keeping animals created specifically as living machines for the production of proteins, antibodies, etc.