SEARCH

SEARCH BY CITATION

Keywords:

  • avian;
  • germ cells;
  • transgenesis;
  • bioreactor;
  • biomodel

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. History of avian models
  5. Advantages of avian models
  6. Technological advances in the production of transgenic birds
  7. Future prospects for avian models
  8. Conclusions
  9. Acknowledgments
  10. Conflicts of interest
  11. References

Animal-based biotechnologies involve the use of domestic animals for the production of pharmaceuticals and various proteins in milk and eggs, as disease models, as tools for stem cell research and animal cloning, and as sources of organs for xenotransplantation into humans. Avian species offer several advantages over mammalian models, and they have been used historically to advance the fields of embryology, immunology, oncology, virology, and vaccine development. In addition, avian species can be used for studying the etiology of human ovarian cancer and other human diseases such as disorders based on the abnormal metabolism of lipids and as unique mechanisms for the biosynthesis and transport of cholesterol. This review integrates recent progress and insight into the molecular and physiologic mechanisms associated with transgenic birds and gives an overview of the use of avian models as pharmaceutical bioreactors and as tools for studying human diseases.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. History of avian models
  5. Advantages of avian models
  6. Technological advances in the production of transgenic birds
  7. Future prospects for avian models
  8. Conclusions
  9. Acknowledgments
  10. Conflicts of interest
  11. References

Substantial efforts have been made to overcome disease and to enhance the quality of human life through advances in medicine and biotechnologies. Since the completion of the human genome project, there has been intense interest in mining the human genome database for genomic signatures of disease for diagnosis and drug development. Many researchers use animal models to understand human disease, and the results of these studies have helped to reduce morbidity and mortality. An animal model can be defined as a living organism that is closely related phylogenetically to humans and that can be used to study shared biological systems in relation to health and disease. Thus, the genetic similarities among animals in a taxonomic system are validated on the bases of comparative DNA and RNA analyses. A range of approaches are needed to develop and refine animal models for transgenic applications. Currently, transgenic animals are valuable models for assessing therapeutics for humans. Animal disease models that allow long-term assessment of alternative therapies are also necessary. This is especially true for animal models that are developed or customized to study specific diseases, so as to elucidate the biological pathways implicated in those diseases. Although rodent models have proven useful in the past in revealing disease etiology and developing treatments, avian models are emerging as important alternatives. This review presents an integrated view of recent progress and new insights into the molecular and physiologic mechanisms that are relevant to the use of avian models as bioreactors for the production of therapeutics and for studies of human diseases.

History of avian models

  1. Top of page
  2. Abstract
  3. Introduction
  4. History of avian models
  5. Advantages of avian models
  6. Technological advances in the production of transgenic birds
  7. Future prospects for avian models
  8. Conclusions
  9. Acknowledgments
  10. Conflicts of interest
  11. References

Avian embryos have been used for many basic and translational studies because they develop in ovo independently of most environmental factors. Aristotle used chick embryos to monitor organ development through various stages and concluded that life is generated spontaneously. William Harvey monitored the formation of arteries, veins, capillaries, and vessels in developing chick embryos. In the early 1800s, Malphigi paved the way for studies of animal development by monitoring neural tube and somite formation in chick embryos.1 In 1911, Rous first demonstrated that tumors could be induced by a virus (thereafter called Rous sarcoma virus; RSV), and Temin discovered the retrovirus and, subsequently, the function of reverse transcriptase in 1970. These advances led to the establishment of major concepts in modern virology.2 In addition, the avian immune system has been used for studying the differentiation and function of B and T lymphocytes. Chick embryos are commonly used to study body axis formation, neurogenesis, limb generation, ocular development, and muscle differentiation.1 Chick embryos are also potential research models for studies of oncogenesis, viral disease, vaccine development, and human diseases, as well as for the development and testing of therapeutic agents. The full potential of avian species as research models will be realized through studies in the agricultural, biomedical, and industrial fields.

Advantages of avian models

  1. Top of page
  2. Abstract
  3. Introduction
  4. History of avian models
  5. Advantages of avian models
  6. Technological advances in the production of transgenic birds
  7. Future prospects for avian models
  8. Conclusions
  9. Acknowledgments
  10. Conflicts of interest
  11. References

Avian embryos have many advantages as a research model, because they develop independent of maternal influences, thereby facilitating multiple experimental procedures in ovo. Manipulations of postgastrulation stage embryos (e.g., isolation, transplantation, and gene transfer into various cells) allow experimental procedures and various types of analyses of living cells. Avian embryos also permit monitoring of organogenesis, because the distinct characteristics of development can be observed as the embryonic organs form and develop.

The estimated total number of genes in the chicken is similar to that in humans, although the chick genome is only half the size of the human genome. The chicken genome was the first nonvertebrate amniote genome to be sequenced.3 Therefore, recent results regarding the evolutionary correlations between humans and chickens provide information on the general aspects of the evolution of animal genomes.

Avian models are appropriate for research in the basic scientific disciplines. Moreover, these models can be further utilized for biotechnological and agricultural applications. Birds have well-developed, economically important traits, such as egg laying, weight gain, and lipid metabolism. Furthermore, the genetic pathways in chickens that regulate these traits may be used in human research to, for example, identify the etiologies of cardiovascular disease, obesity, and chronic wasting syndromes.4 In the chicken, reproductive traits are controlled by photoperiodicity, and a laying hen can produce more than 300 eggs annually. Moreover, the chicken is regarded as an optimal bioreactor system owing to its many biologically active nutrients and the fact that the egg proteins are structurally simple.5,6 Because hens ovulate almost daily and are highly susceptible to developing ovarian cancer once the egg-laying period is finished, they are more valuable for studying human ovarian cancer than are rodent or human models, in which ovulation is less frequent or must be stimulated.7 Moreover, many hens can be maintained and managed in a limited amount of space, and one can expect to find various lesions from hens with ovarian cancer or other diseases.

Technological advances in the production of transgenic birds

  1. Top of page
  2. Abstract
  3. Introduction
  4. History of avian models
  5. Advantages of avian models
  6. Technological advances in the production of transgenic birds
  7. Future prospects for avian models
  8. Conclusions
  9. Acknowledgments
  10. Conflicts of interest
  11. References

The transfer of a gene construct into the pronucleus of a zygote is the most commonly used method for producing transgenic mammals. After the production of the first transgenic mice, pronuclear injection became the method of choice for producing transgenic animals.8 Spurred on by the accomplishments in mammals, similar efforts to produce transgenic birds in the mid-1990s revealed that pronuclear injection was not feasible in avian zygotes.9,10 Pronuclear injection in avian species is technologically demanding because the rapid development of the embryo hinders the selection of suitable embryos for gene insertion. In addition, it is not easy to collect zygotes from the hen oviduct using surgical procedures, and the presence of a nontranslucent yolk surrounding the embryos interferes with the precise positioning of the pipette tip to deliver the gene constructs into the zygote. The most efficient method for producing transgenic avian species is to introduce retroviral gene constructs into the embryos immediately after oviposition, i.e., the laying of the egg.

The embryos of freshly laid eggs have two embryonic layers that contain 40,000–60,000 undifferentiated cells. After genes are introduced into these structures, they are transduced into the somatic and germ cells and a genetically mosaic chick is produced. This bird is bred at sexual maturity to produce a mosaic bird that is crossed with its wild-type counterpart to obtain G1 transgenic offspring. It has been demonstrated that retroviral constructs transferred into embryos at this stage create transgenic germ cells.11 More recently, the production of transgenic chickens that carry the β-lactamase gene in their genomes was accomplished using avian leukosis virus (AVL)-based retroviral vectors.12 Remarkably, the gene construct was successfully transmitted to the next generation without altering expression of the target gene. That study was the first to indicate the feasibility of producing biopharmaceuticals in a hen bioreactor system. Thereafter, various retroviral vector systems were developed. However, it is now clear that the expression of target genes transferred by retroviral vectors can be altered in various tissues and at various developmental stages.13,14 To overcome these limitations, multiple classes of lentiviral vectors are used to produce transgenic birds (Fig. 1).15–18 An alternative method is to transfer the retroviral concentrates into the circulation of chick embryos incubated for 53h ∼ 55h to prevent gene silencing via a mechanism that involves transgene variegation but that is currently not well defined.19

image

Figure 1. Schematic of the production of transgenic birds. (A) Production of transgenic birds by lentiviral transduction of stage X embryos. (B) Production of transgenic birds using lentivirus-transduced primordial germ cells (adapted from Shin et al.18).

Download figure to PowerPoint

Despite the many technological advances and several failures, the search continues for new methods to produce transgenic birds. Some major obstacles need to be overcome. First, the retroviral construct integrates randomly into the host chromosome, which can lead to transgene expression at various developmental stages and in various tissues. This is caused by neighboring chromosomal elements that act as negative regulators of transgene expression. Such epigenetically regulated phenomena are called “position effects” and may be overcome by including additional regulatory elements in the delivered transgene construct. Second, the rate of germline transmission of proviral constructs can be quite low, thereby complicating the process of establishing transgenic lines in birds. Third, proviral constructs can become oncogenic due to the presence of innate retroviral elements.

Primordial germ cells (PGCs) are the precursors of functional sperm and oocytes. PGCs have unique translocation and migration abilities during embryonic development and they ultimately colonize the urogenital ridge of the embryo. The PGCs in birds originate from the epiblasts and translocate into the hypoblast during incubation. After approximately 16 h, the PGCs congregate in the anterior extra-embryonic area called the “germinal crescent.” Thereafter, with the development of the embryonic cardiovascular system, the PGCs migrate, enter into the circulation, and eventually colonize the urogenital ridges. Exploiting this unique developmental sequence, the transfer of PGCs from donor to recipient embryos results in the production of germline chimeras, and ultimately, transgenic chicks are derived from the retrovirally transduced PGCs.20,21 However, the usefulness of PGCs as a vehicle for gene transfer is limited by the low number of PGCs that can be extracted from the germinal crescent and that are suitable for gene transfer. Attempts have been made to overcome these limitations by using cultured PGCs. Embryonic germ cells are pluripotent stem cells that are derived from PGCs through an extended culturing period.22 Park and Han were the first to succeed in producing embryonic germ cells by culturing PGCs harvested from embryonic gonads.23 They demonstrated that these cells differentiate into three germ layers, the origins of which are developmentally distinct. These pluripotent cells can be used to create chimeric recipient embryos, and they differentiate into gametes.24 It is noteworthy that PGCs that were cultured, transfected, and transferred to a recipient embryo successfully generated a transgenic chick and a transgenic quail (Fig. 1).18,25

Because avian PGCs are transported via the blood vessels during embryonic growth, they colonize the urogenital ridges and give rise to germline chimeras in birds, as a means of conserving the species in ecosystems. Currently, there are hundreds of species of birds at risk of extinction. The generation of germline chimeric poultry by transferring PGCs from endangered bird species is an ideal method for restoring depleted populations of bird species. This idea has been validated by the reported production of viable pheasants using chickens, which demonstrates that this technique can be used to produce wild birds via germ cell transplantation.26

Tissue sources other than embryonic blood or gonads can also be used for generating germline chimeric birds. Spermatogonial stem cells (SSCs) are progenitors of sperm that reside on the peripheral boundaries of Sertoli cells. SSCs give rise to differentiated spermatogonial cells, plus an identical SSC as a mechanism for self-renewal. In chickens, PGCs are collected from early embryos (embryos from 2.5 to 5.5 days), whereas SSCs are harvested from the differentiated testes of male chicks that are several weeks old. SSCs can be used to produce donor-derived offspring, because high numbers of cells can be harvested and there is no requirement to wait until the male recipient chicks become sexually mature. One of our research interests includes developing a novel method for reproduction in birds.27 Given the potential of SSCs to give rise to germline stem cells (GSCs), the production of transgenic offspring from SSCs would replace the conventional embryo-mediated transgenesis systems developed during the last few decades.28 Such efforts will be helpful for studying the mechanisms that control pluripotency and dedifferentiation in germ cells.

Future prospects for avian models

  1. Top of page
  2. Abstract
  3. Introduction
  4. History of avian models
  5. Advantages of avian models
  6. Technological advances in the production of transgenic birds
  7. Future prospects for avian models
  8. Conclusions
  9. Acknowledgments
  10. Conflicts of interest
  11. References

Technologies for producing transgenic birds have evolved significantly over the last few decades, although the potential applications have not been thoroughly explored. A self-contained avian embryo undergoing development offers numerous advantages as an animal biomodel.29 The chick is free from maternal effects, which allows accurate assessments of the responses to various treatments, including environmental chemicals and physical stress. This unique in vitro-like—but in fact in vivo—system allows the monitoring of developmental events over time via eggshell windows, and the use of well-established surrogate systems for incubating embryos so that they have high rates of survival and normal developmental patterns. This classical embryologic model has been used to study developmental processes such as aging as well as hematopoiesis and the development of muscles and neurons.30–32 The chicken is also a good model for studying human diseases such as lymphoma and for regenerative medicine.33,34 Chickens are especially valuable models of ovarian cancer due to their unique physiologic traits.35–39 The murine model is not suitable for studying this disease because, in the mouse, ovarian cancer originates mainly from granulosa cells or germ cells and not from the epithelium of the ovary.35 Although epithelial ovarian cancer can be artificially induced by carcinogens, the overexpression of the SV40 large T antigen, or through the use of xenografts, these modes do not support efforts to identify the etiology of human epithelial ovarian cancer.40 The physiologic characteristics of epithelial ovarian cancer in chickens are similar to those of the cancer in humans. Convincing evidence exists that human epithelial ovarian cancer results from the accumulation of DNA damage in the ovarian epithelium due to incessant ovulation.41 In humans, the incidence of epithelial ovarian cancer tends to increase dramatically after menopause or when the regular cycles of ovulation cease due to the hormones of pregnancy or the use of oral contraceptives.41 The chicken is an excellent model for studying epithelial ovarian cancer because hens ovulate almost daily and produce about 300 eggs annually. Other studies have reported that continuous DNA damage occurs in the ovarian epithelial cells while the laying hen produces eggs, which is consistent with the major risk factors for human epithelial ovarian cancer.42 In conjunction with the established transgenic techniques, avian biomodeling systems will advance the development of human disease models and will promote research approaches to improve human health and well-being.

Eggs contain lipids, proteins, carbohydrates, minerals, and other nutrients, and can be used in cooking and in further processing. Although eggs have clear nutritional value, persons who are suffering from atherosclerosis or who have a family history of hyperlipidemia may need to limit their intake of eggs, which contain high levels of cholesterol. To reduce the cholesterol content of eggs, hens are managed so as to modify their lipid sources, e.g., through fiber-rich feed or by adjusting the intake of micronutrients. However, such efforts are not sustainable because they are not cost-effective for poultry operations engaged in the large-scale production of eggs. In hens, cholesterol is synthesized in the liver and transported to peripheral tissues, including the ovary, in the form of very low-density lipoproteins.43 Several key regulators of cholesterol synthesis have been identified, and it seems likely that the lipid content of eggs can be reduced.

Major pharmaceutical companies have concentrated on developing novel drugs because the potential for success is greater than for other applications. The overall market value of recombinant proteins was US$18.8 billion in 1997, and it is expected to be more than US$57 billion by 2010. However, major technological advances have been made using Escherichia coli, insect cells, fungi, yeast, and mammalian cells to produce therapeutic molecules that are subsequently used in translational or clinical studies of human disease. Conventional cell culturing systems entail high maintenance costs, and the products are often not suitable for therapeutic purposes due to the lack of appropriate structural modifications. Opportunities for the mass production of biopharmaceuticals in egg-white proteins are significant. When this becomes feasible, the pharmaceutical industry will undergo a revolutionary change.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. History of avian models
  5. Advantages of avian models
  6. Technological advances in the production of transgenic birds
  7. Future prospects for avian models
  8. Conclusions
  9. Acknowledgments
  10. Conflicts of interest
  11. References

In this review, we have focused on the roles of transgenic avian species in the past, as well as on the current procedures in manufacturing and biotechnology to produce products of importance to the agricultural, academic, and industrial sectors. Avian models are currently being used to study human diseases and to assess the efficacies of candidate therapeutics. Considering that the major concepts underlying studies of aging, stress responses, cell differentiation and dedifferentiation, and metabolic syndrome have been established in lower vertebrates, the use of avian models is expected to expand and intensify as efforts are made to elucidate the physiologic states of health and disease. Furthermore, avian models can be developed for industrial purposes to produce human proteins, therapeutics, and foodstuffs. Future studies are expected to result in the development of advanced transgenic technologies that exploit and enhance the advantages of avian models for basic and translational research.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. History of avian models
  5. Advantages of avian models
  6. Technological advances in the production of transgenic birds
  7. Future prospects for avian models
  8. Conclusions
  9. Acknowledgments
  10. Conflicts of interest
  11. References

This research was funded by the World Class University (WCU) program (R31-10056) through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology, and by a grant from the Next-Generation BioGreen 21 Program, Rural Development Administration, Republic of Korea.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. History of avian models
  5. Advantages of avian models
  6. Technological advances in the production of transgenic birds
  7. Future prospects for avian models
  8. Conclusions
  9. Acknowledgments
  10. Conflicts of interest
  11. References