Avian Transgenic: An industrial perspective

Sandeep Goel and Satish Kumar
Transgenic and Gene Knockout Mice Laboratory,Centre for Cellular and Molecular Biology,Uppal Road, Hyderabad - 500007
Email: [email protected]

Genetic modification of animals was initiated in 1980s and now it is possible to modify many livestock species, enabling development of transgenic mammals with numerous commercial applications (Clark et al., 1987, Pursel 1989, Denman et al., 1991, Wright et al., 1991, Velander et al., 1992). Several potential applications of transgenic chickens have been identified and the two main areas include agriculture and healthcare. The chicken, in theory, is fantastic production vessel for recombinant proteins, because hens are nature’s most efficient bioreactors. A modern layer lays approximately 300 eggs an year and the egg white alone contains four grams of protein. If a mere ten milligram of this is recombinant human protein, it will be enough for efficient scale up. Moreover, poultry production is a well-organized industry. These advantages suggest that estimated cost of production of recombinant protein in an egg would be half of that produced by state-of-art bioreactor growing engineered E coli or Chinese Hamster Ovary (CHO) cells (Houdebine 2000). Hen takes much less time to reach sexual maturity than any other livestock species. Another major advantage of chicken egg is that post-translational modifications of protein are similar to those of human (Raju et al., 2000). Therefore, transgenic chicken holds a tremendous potential to revolutionize the biotechnology industry and would contribute significantly to the economy of the country. However, development of a robust method for production of transgenic chickens has been more of a challenge.

Methods of Genetic Modification in Chickens

Direct microinjection of DNA

Unlike livestock and mice, chicken eggs are difficult to work with. Conventional method of genetic modification by microinjection of DNA into oocytes established for mammalian species (Palmiter et al., 1982, Clarke et al., 1987) has not been easy to work with in chicken. This is because of the complex process by which a hen produces and lays fertilized eggs. Unlike mammalian eggs, pronuclei of chicken egg are difficult to visualise as they lie 22-25 m below the vitalline membrane and cytoplasm is opaque due to presence of white yolk particles ( Waddington et al., 1998). Moreover, egg laying takes ~ 22 hours and by that time the embryo has already developed as a disc of ~ 60,000 cells overlaying the germinal cavity (Spratt and Haas, 1960). To harvest ovum for pronuclei injection, a hen has to be sacrificed for each ovum and ex-vivo ovum culture is required which have low hatchability (Perry 1988, Sherman et al., 1998). Germline transgenic cockerel was first produced by microinjection of linearised gene construct by Love et al., 1994. This technique has not gained much popularity due to low rate of success and complexity of this procedure in chicken.

b) Viral methods

Gene transfer into the avian genome was first achieved using replication-competent vectors derived from avian leucosis virus (ALV), an onco retrovirus, by transduction of stage X blastoderm (Salter et al., 1986, 1987). This method proved to be inefficient as the frequency of production of germ line chimera was very low and expression of transgene carried by these vectors was quite poor. Moreover, this procedure was commercially unsuitable since transgenic birds shredded live viruses. Replication deficient retroviral system was developed and this also faced problem of low efficiency of transgenesis (Bosselman et al., 1989). Recently, novel vectors have been derived from the lentiviral group of retroviruses. These vectors have several advantage over those derived from onco retroviruses, including the ability to infect non-dividing cells (Buchschacher et al., 2000). These vectors have the capacity to carry larger transgene (up to 10kb) and have been shown to be compatible with tissue-specific transgene expression after germ line transmission in transgenic mice (Lois et al., 2002; Pfeifer et al., 2002). Transgenic chickens have been generated using lentiviral vector that express GFP and LacZ by McGrew et al., 2004. Use of lentiviral vector has overcome the problem associated with retroviral vectors both in term of transgene expression and frequency of production of transgenic birds (Sang, 2004a).

c) Transposon mediated

Transposons or ‘jumping genes’ are mobile pieces of DNA that can move from one location to another within a genome through transposition. Transposons transposes by ‘cut and paste mechanism’ when injected into cytoplasm of chick zygotes. Use of transposable elements mariner (Sherman et al., 1998) to produce transgenic chicks has been reported. These elements transposes at high frequency into the chicken genome suggesting that mariner or another transposon like ‘Sleeping Beauty’ element (Isvak and Ivics 2004) could be used to improve the efficiency of transgenesis, although there are limitation on the size of transgene these vectors can carry.

d) Sperm mediated

Sperm mediated gene transfer has gained importance as a method of gene delivery into mammals, (Lavitrano et al., 2002/2003). However, there are no data to suggest that a transgene of interest can be successfully incorporated into the avian germline. Integration of foreign gene in sperm is quite difficult as sperm are quiescent cells and DNA is condensed. There are unpublished reports where the foreign gene tagged to monoclonal antibody against sperm antigen has been used to deliver into sperm.

 Chicken Stem Cells

Another technology to create transgenic chicken is to use genetically modified blastodermal cells and primodial germ cells (PGCs). Blastodermal cells are pluripotent cells, which contributes to most embryonic cell lineages including those giving rise to germ line (Petitte et al., 1990). The manipulated blastodermal cells are implanted into the developing embryo to create a germ line chimera. On the other hand PGCs arise from the central region of blastoderm that circulate in embryonic blood and colonise in genital ridge, which eventually differentiate into oocytes and spermatocytes. PGCs can be isolated directly from embryonic blood; the developing gonad or the germinal ridge. Manipulated PGCs can then be injected into embryonic blood stream where they migrate to the germinal ridge. The cell-based method of gene insertion is attractive because the integration into the genome and sometimes expression of the transgene can be examined before the manipulated cells are implanted into the embryo. Further, it allows introduction of precise mutation into specific genes. Production of transgenic chicken stem cells comprises of three components 1) The ability to culture cells capable of entering the germ line 2) The development of suitable DNA constructs and genetic modification of cells in culture 3) The ability to produce germ line chimeras from manipulated cells. Petitte et al., (1990) produced first chicken germ line chimeras using stage X blastodermal cells. There is no report as yet that chick blastodermal cells that have been maintained in culture for more than a week can contribute to the germ line. Significant advances have been made in production of germ line chimeras by transfer of PGCs from one embryo to another and it is now possible to maintain germ line competent PGCs in culture up to 90 days (Park et al., 2003a and 2003b). Successful germ line transmission of modified cells has not been reported (Petitte et al., 2004). Development of cell culture protocols that would permit growth and maintenance of PGCs and blastodermal cells over extended period in vitro without compromising the germ line competence would revolutionize transgenesis in chicken.

Transgene Design

In developing the hen as a bioreactor, it is important to show that a transgene can be expressed in egg white. To achieve this, a transgene will have to be driven by oviduct specific promoter. More than half of the egg white protein is product of ovalbumin gene. The other major proteins of egg are lysozyme, ovomucoid, ovamucin and conalbumin. (Gilbert et al.,1984). Unlike mammary gland promoters, which were tested for specificity and activity in transgenic mice before committing to the expense of transgenic livestock, birds lack this luxury. Ovalbumin promoter has been analyzed extensively in explant culture of chick oviducts (Sensenbaugh and Sanders, 1999). Lysozyme gene promoter is next widely studied (Bonifer et al., 1997). Other egg white gene promoters remain largely unexplored. Apart from promoter, other factors like hormone (e.g. estrogen) and cis elements that control gene expression need to be examined extensively to understand their role in transgene expression (Ivarie, 2003).

Potential Applications

Chicken eggs have been used in the manufacture of vaccines for more than 30 years. Given the existing poultry and vaccine industry infrastructure for pathogen-free egg production and pharmaceutical grade product processing, and the inherent efficiencies of chicken as protein producers, transgenic chicken can outperform other recombinant biopharmaceutical production system due to short time-to-market, low production and capital costs and safe production (Sang, 2004b). Improving agronomic traits, such as growth and disease-resistance, can quickly be incorporated into commercial poultry breeding and growing programs, revolutionizing the economics of the poultry productions. Some of the specific applications are listed below:

  1. Enhancement of short-term, broad-range diseases resistance in chicken.
  2. Targeted expression of gene enhancing muscle fiber hyperplasia and hypertrophy and increase in dark and white muscle through gene targeting.
  3. Introduction of unique signature DNA sequences to protect proprietary rights to established breeder and newly developed transgenic line, effectively acting as genetic encryption device.
  4. Production of recombinant feed supplements and growth-promoting antibiotics in poultry eggs for feeding farm animals to increase their growth rates.
  5. Production of monoclonal antibodies for therapeutic use.
  6. Produce eggs with lower cholesterol for human consumption
  7. Produce recombinant vaccine, hormones and therapeutic proteins in egg (Rapp et al., 2003)
  8. Fortify egg with proteins e.g. soy-isoflavons to improve the biological value of egg.
  9. Produce yolk antibodies like avian immunoglobulin (IgY) for diagnostics substituting use of laboratory mammals.
  10. Use cloning technology to mass-produce broilers in eggs of flock of high producers hens e.g. white leghorn.
  11. Produce broilers expressing fat reducing gene and expressing growth promoting hormones making it possible to produce birds which are larger, leaner and with faster growth rate.
  12. Producing transgenic chicken expressing various digestive enzymes in their gut enabling efficient digestion of feed which would increasing the feed conversion efficiency e.g. galactosidase that facilitate lactose digestion (Mozdziak et al., 2003).

Use of RNA interference (RNAi) in chicken is likely to generate novel approaches for modification of chicken genome. RNAi is an exciting new technology that involves the use of small interfering double stranded RNA (siRNA). RNAi is a protective innate response observed in a wide variety of organisms, and is best considered a feature of all eukaryotes. At the cellular level, the detection of a double stranded RNA molecule triggers the specific degradation of the mRNA of an exact sequence to an expressed gene, leading to loss of phenotype (Fire et al. 1991). This technology has been developed as the method of choice to produce gene “knockdowns” for high throughput functional genomic studies in a number of species. RNAi has potential for therapeutic applications as well (Caplen 2004). Control of infection, in particular, persistent viral infections such as HIV is being heavily studied. siRNA molecules against essential viral genes have been shown to reduce viral loads in cell culture. Gene specific therapeutics for cancer and auto-immune diseases are also being investigated and these show promise in in vitro systems. A major obstacle to overcome in the development of RNAi therapeutics is efficient delivery of siRNA’s into target cells in vivo. The advantages of lentivirus vector for gene delivery would bridge this gap and would make a revolutionary change in genetic modification of chicken with more efficiency and cost effective. There are reports of use of RNAi as a tool to investigate gene function both in chickens and Eimeria as to develop RNAi as a novel therapeutic for control of coccidiosis. RNAi can be used to control expression of genes with important production benefits in particular genes controlling sex differentiation and regulating muscle development. This technology can be further extended to combat major viral diseases like Marek’s and IBD.

In both the broiler and layer industries, priority is given to improving immuno-responsiveness and resistance to a variety of avian diseases. To improve disease resistance it might be necessary to attack pathogens at more than one point in their life cycle because any single gene change is likely to be overcome quickly by pathogen mutations.

Avian Transgenic Companies

Recently, many private research companies have initiated research in the field of avian transgenics to harness this powerful emerging technology. Some of the companies listed are:

Companies Main approach to
transformation of chicken
Advanced Cell Technology, Worcester, MA Avian embryonic germ cell
AviGenics,Athens, GA Retroviral-mediated; other methods?
BioAgri, City of Industry, CA Sperm-mediated transgenesis
GeneWorks, Ann Arbor, MI Retroviral mediated; some stem cell work
Origen Therapeutics, Burlingame, CA Avian embryonic stem cell
TranXenoGen, Shrewsbury, MA Direct egg transfection; other methods
Viragen, Plantation, FL Confidential, Transposons??
Vivalis, Nantes, France Avian embryonic stem cell

These companies have been using various methods of genetic modifications motioned in this paper. Some of the companies do not disclose the methods used by them. Presence of large numbers of companies, at least, proves that transgenic poultry holds a lot of promise and a major breakthrough in transgenic technology enabling easy modification of poultry genome would flag off the race in this field.

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