Jake Harper
Chickens are genetically modified organisms (GMOs) by the USDA's definition. Genetic modification can occur through selective breeding or gene splicing. The latter is a rare practice in commercial animal farming and involves changing an organism's genetic makeup by introducing, eliminating, or rearranging specific genes. Transgenic chickens have been generated over the last two decades via viral and non-viral transfection. Viral vectors are the most successful method, as the DNA construct naturally integrates into the host chromosomes. However, drawbacks include the replication of deficient viral particles and the risk of recombination with wild-type viruses. Non-viral methods involve genetically modifying embryonic cells and transferring them into the recipient embryo. The production of transgenic birds has been challenging due to the unique structure of the chicken zygote and embryo.
| Characteristics | Values |
|---|---|
| First genetically modified chicken | 1989 |
| Techniques | Viral transfection systems, non-viral methods, gene splicing, direct DNA microinjection, viral vector transfer, cloning of transfected nuclei, plasmid-DNA microinjection, lentiviral vectors |
| Applications | Medicine, drug manufacturing, developmental studies, research, investigation of disease susceptibility, creating biomedical models, recombinant protein production, biopharming, production of therapeutic recombinant proteins |
| Benefits | Reduced production costs, increased efficiency, superior to transgenic plants and insects, lower risk of adverse immune responses to drug proteins, higher biological activity |
| Concerns | Zoonotic diseases, welfare concerns for mother hens, risk of fractures, ethical concerns |
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What You'll Learn
Viral transfection systems
Transgenic chickens have been produced using viral transfection systems. These systems are among the simplest biological agents capable of transferring genes into the central nervous system. Viral vectors are used to transfer exogenous genes into various cell types, including neurons and glial cells.
Several commonly used viruses for viral transfection include adenoviruses, adeno-associated viruses (AAVs), and lentiviruses. Adenoviruses have a high packaging capacity and can transduce most cell types. AAVs, on the other hand, have lower immunogenicity and pathogenicity, making them safer for gene therapy. Lentiviruses are also advantageous due to their simplicity, low pathogenicity, and ease of production.
When choosing a viral vector for transfection, factors such as the type of transfected nucleic acids (DNA, RNA, or small RNAs) and the complexity of the transfection procedure must be considered. Additionally, the choice of a suitable transfection reagent is important. For example, Effectene and TransIT-X2 are reagents specifically designed for plasmid DNA transfection, while Lipofectamine RNAiMAX is more suitable for small oligonucleotide transfection.
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Non-viral methods
Transgenic chickens have been produced by two different procedures, the first being based on viral transfection systems, and the second being a non-viral method. This method involves genetically modifying embryonic cells that are then transferred directly into the recipient embryo.
There are various non-viral methods to create transgenic chickens. One way is to inject a vector into the vascular system or directly into the heart of developing embryos after 50 to 60 hours of incubation. This method was used by Tajima et al. to generate the first chimeric chicken by transplanting 100 chicken PGCs into a recipient embryo. However, this process is difficult because it creates a mosaic of PGCs in which only a small portion are transfected.
Another non-viral method is to inject the Tol2 plasmid-liposome complex into the early embryonic dorsal aorta, which was found to improve the rate of producing G0 transgenic roosters.
Furthermore, direct DNA microinjection into the pronucleus is a frequently applied technique in mammals, but it is almost impossible in birds. This is because fertilization in hens occurs in the infundibulum of the reproductive tract, and it is difficult to identify the male pronucleus among the supernumerary spermatozoa.
One promising model for the creation of transgenic birds is the culture of chicken oviduct epithelial cells (COEC) that can be easily transfected with constructs based on oviduct-specific regulatory sequences.
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Genetic engineering
Chickens have been genetically engineered for various purposes, including the production of therapeutic recombinant proteins, which are crucial for drug development and medicine. The first genetically modified chicken was reported in 1989, and since then, many other transgenic avian species have been generated, with a focus on chickens and quails.
One of the main advantages of using chickens as bioreactors for protein-based drugs is their ability to produce complex and active proteins with the appropriate post-translational modifications. This is a challenge for bacterial bioreactors, which is the most cost-effective system. Transgenic chickens can produce proteins in their egg whites, which can be easily harvested for purification. The ovalbumin promoter facilitates the localized production of ovalbumin, the main protein in egg whites, and this promoter can be modified to regulate the production of a gene of interest (GOI).
There have been two main procedures used to generate transgenic chickens: viral transfection systems and non-viral methods. The viral transfection method involves the insertion of retroviral foreign DNA, usually delivered by an avian leukosis virus, into the germline. Various viral vectors have been used, but they come with drawbacks such as the replication of deficient viral particles and the risk of recombination with wild-type viruses. The non-viral method involves genetically modifying embryonic cells and directly transferring them into the recipient embryo. This method includes several steps such as isolation and cultivation of donor embryonic cells, transgene construction, cell transfection in vitro, and chimera production.
Overall, the genetic engineering of chickens has opened up new possibilities for drug development, medicine, and research.
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Bioreactors
Chickens have been transformed into transgenic organisms using bioreactors for the purpose of producing therapeutic recombinant proteins. This is because chickens have the ability to produce complex and active proteins, as well as provide the appropriate posttranslational modifications.
The first genetically modified chicken was reported in 1989, and since then, many other transgenic avian species have been generated. Transgenic chickens are created using bioreactors, which are systems that allow for the production of recombinant proteins in their mammary glands or other secretory organs. The process involves introducing foreign DNA sequences into the genome of transfected cells and ensuring that the DNA sequences are integrated and transmitted to the offspring. The most common methods for creating transgenic chickens include pronuclear microinjection, embryonic stem cell-mediated gene transfer, and retrovirus-mediated gene transfer.
The chicken bioreactor has the potential to reduce the costs and increase the availability of therapeutic proteins for both human and veterinary medicine. This is because the infrastructure and upstream costs of a transgenic chicken bioreactor production system are predicted to be substantially lower than those for mammalian cell culture. In addition, the time and expense needed for chickens to reach maturity and begin producing target compounds are much less than those needed for other farm animals.
The process of creating a transgenic chicken using a bioreactor involves culturing chicken primordial germ cells (PGCs) and their ability for germline transmission. The CRISPR/Cas9 system is then used to integrate a desired gene into a predetermined position in the genome. This system has been successful in modifying the genome of chickens to produce pharmaceutical and nutritional proteins.
Overall, the use of bioreactors to transform chickens into transgenic organisms has the potential to revolutionize the production of therapeutic proteins, making it more cost-effective and efficient.
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CRISPR/Cas9 system
The CRISPR/Cas9 system is a powerful tool for genome editing in various organisms, including livestock animals. It involves the use of the CRISPR/Cas9 nuclease to target specific DNA sequences and generate double-stranded breaks (DSBs) in genomes. This allows for the introduction of exogenous genes or indels into chromosome DNA breaks, resulting in targeted transgenesis or mutagenesis. The system has been applied to chickens to modify their genetic information and create transgenic poultry.
One example of the use of the CRISPR/Cas9 system in chickens is the generation of ovomucoid gene-targeted chickens by Oishi et al. (2016). They transferred transiently drug-selected primordial germ cells (PGCs) into recipient embryos using gamma-ray irradiation to deplete endogenous PGCs. This resulted in the establishment of germline chimeric roosters (G0) with donor-derived mutant-ovomucoid spermatozoa. The offspring of these roosters were heterozygous mutant ovomucoid chickens, indicating successful gene-specific knockout in chickens.
Another study by Veron and his group (2015) used the CRISPR/Cas9 system to create the first CRISPR/Cas9-mediated chicken through electroporation of chicken embryos. They combined an in vitro culture system for PGCs with the efficient genome-editing system to produce programmable genome-edited poultry. This approach has also been used to create knock-in/out chickens, low-allergenicity eggs, and disease-resistant models.
The CRISPR/Cas9 system offers several advantages over other genome editing technologies, such as homologous recombination and the TALEN method. It is a simple and highly efficient method for gene targeting, requiring only simple plasmid construction. It also enables the understanding of gene function and the modification of the phenotype of animals according to defined production and scientific goals.
However, one potential disadvantage of the CRISPR/Cas9 system is the possibility of off-target effects. While some studies have reported frequent off-target mutagenic effects, others have found that off-target mutations are rare and not a serious problem for CRISPR/Cas9-mediated genome editing. Additional analyses may be required to precisely evaluate the off-target effects associated with mutagenesis by CRISPR/Cas9 in chickens.
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Frequently asked questions
Transgenic chickens are chickens that have been genetically modified, often to serve as bioreactors for the production of recombinant proteins that are crucial for therapeutic applications.
Transgenic chickens have traditionally been produced by two different procedures. The first procedure is based on viral transfection systems. The second procedure, the non-viral method, is based on genetically modified embryonic cells transferred directly into the recipient embryo. Other methods include direct DNA microinjection into the pronucleus and cloning of transfected nuclei transferred into enucleated oocytes.
Transgenic chickens can be used as powerful bioreactors to produce pharmaceutical and nutritional proteins. They can also be used to produce recombinant proteins that are crucial for therapeutic applications.
According to the National Chicken Council, there are no food safety or other risks to the health and well-being of consumers when they consume chicken or other animal agriculture products (e.g. eggs, dairy) that have been raised with genetically modified feed ingredients. This position is supported by the World Health Organization (WHO).
