Transgenic Chickens Research Articles

Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan

The transgenic chicken is a candidate for the production of biopharmaceutical proteins with several economic superiorities. In general, the addition of sialic acid at the terminal of N-glycan is important for the bioactivity of biopharmaceuticals including plasma half-life; however, sialic acid has not been detected in the N-glycan of proteins produced in the egg white of genetically manipulated chickens. In this study, the extracellular domain of the TNF receptor and single chain Fv fused to Fc (referred to as TNFR/Fc and scFv/Fc, respectively) were purified from the egg yolk of genetically manipulated chickens and their sialylation in N-glycan was examined. In contrast to the glycan in egg white, yolk-derived proteins were partly sialylated. Lectin blot showed the existence of α2,6-sialic acid on TNFR/Fc, which disappeared with the removal of N-glycan by PNGase. In scFv/Fc, up to 7% of N-glycan contained sialic acid. Disialyl glycans, which were detected in serum-derived scFv/Fc in a previous study, were not found in the yolk sample. Ovarian follicular tissue, which surrounds growing yolk, expressed several neuraminidases, suggesting the partial truncation of glycan during the yolk transfer process from the blood.

Transgenic Chicken, Mice, Cattle, and Pig Embryos by Somatic Cell Nuclear Transfer into Pig Oocytes

This study explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Enhanced green florescent protein (EGFP)-expressing donor cells were used for the nuclear transfer. Results showed that the occurrence of first cleavage did not differ significantly when pig, cattle, mice, or chicken cells were used as donor nuclei (p>0.05). However, the rate of blastocyst formation was significantly higher in pig (14.9±2.1%; p<0.05) SCNT embryos than in cattle (6.3±2.5%), mice (4.2±1.4%), or chicken (5.1±2.4%) iSCNT embryos. The iSCNT embryos also contained a significantly less number of cells per blastocyst than those of SCNT pig embryos (p<0.05). All (100%) iSCNT embryos expressed the EGFP gene, as evidenced by the green florescence under ultraviolet (UV) illumination. Microinjection of purified mitochondria from cattle somatic cells into pig oocytes did not have any adverse effect on their postfertilization in vitro development and embryo quality (p>0.05). Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Taken together, these data suggest that enucleated pig oocytes may be used as a universal cytoplast for production of transgenic cattle, mice, and chicken embryos by iSCNT. Furthermore, xenogenic transfer of mitochondria to the recipient cytoplast may not be the cause for poor embryonic development of cattle-pig iSCNT embryos.

A new method for producing transgenic birds via direct in vivo transfection of primordial germ cells

Traditional methods of avian transgenesis involve complex manipulations involving either retroviral infection of blastoderms or the ex vivo manipulation of primordial germ cells (PGCs) followed by injection of the cells back into a recipient embryo. Unlike in mammalian systems, avian embryonic PGCs undergo a migration through the vasculature on their path to the gonad where they become the sperm or ova producing cells. In a development which simplifies the procedure of creating transgenic chickens we have shown that PGCs are directly transfectable in vivo using commonly available transfection reagents. We used Lipofectamine 2000 complexed with Tol2 transposon and transposase plasmids to stably transform PGCs in vivo generating transgenic offspring that express a reporter gene carried in the transposon. The process has been shown to be highly effective and as robust as the other methods used to create germ-line transgenic chickens while substantially reducing time, infrastructure and reagents required. The method described here defines a simple direct approach for transgenic chicken production, allowing researchers without extensive PGC culturing facilities or skills with retroviruses to produce transgenic chickens for wide-ranging applications in research, biotechnology and agriculture.

Isolation, Culture and Characterization of Chicken Primordial Germ Cells

Nowadays, production of recombinant proteins in eukaryotes is gaining good deal of attention. Transgenic chicken as a eukaryotic system has a high potential for producing recombinant proteins. Post-translational changes, especially glycosylation, are characteristic of the eukaryotic proteins. In practice we need to choose a proper expressing host when considering over-expression of a recombinant protein. Chickens are among the well-considered candidates for such application. Production of transgenic chickens could be achieved in different ways, including application of primordial germ cells. Primordial germ cells are progenitor of sperm and ovum. These cells are round, with a big nucleus and a cytoplasm with lipid and glycogen particles. The first step for having transgenic chickens is isolation and culture of the primordial gem cells. In the present study, these cells were isolated by centrifugation method in presence of ficoll and using magnetic cell sorting, and were cultured in optimal culture medium. These cells were finally characterized with defined methods, like Periodic acid-schiff staining, alkaline phosphates activity assessment, and antibody staining.

The development of transgenic chickens by sperm-mediated gene transfer

An experiment was carried out to develop transgenic chickens using sperm incubated with exogenous DNA. Two random bred lines of chickens were used as parental flocks, and the plasmid pUC18 was used as the exogene. Lipofectin was also used to facilitate the sperm cell uptake of the plasmid. Hens were inseminated with the semen that was subjected to different treatments and their eggs were hatched. Polymerase chain reaction (PCR) was performed on spermatozoa and blood genomes of the parental flocks and on blood genomes of the progeny. The plasmid DNA was recognized in spermatozoa of both lines in most of the treatments with different amplification rates. However, plasmid DNA was highly fused into the sperm cells when it was introduced in a complex with lipofectin. Also, the plasmid DNA band was highly amplified in the progeny which resulted from sperm cells incubated with the plasmid and lipofectin at 5% concentration. The results reveal the success of the development of F1 chickens by sperm-mediated gene transfer (SMGT). The positively detected SMGT-derived offspring formed 40.0 to 50.0% of the F1 generation. Key words: Rooster spermatozoa, lipofectin, sperm-mediated gene transfer-derived offspring, sperm-mediated gene transfer.

The molecular genetics of avian sex determination and its manipulation

The chicken (Gallus gallus domesticus) has long been a useful model for developmental biologists. The developing avian embryo is easily accessible and fertile eggs are widely available. In addition, the embryo is also amenable to genetic manipulation allowing studies on many important morphological and cellular processes. More recently, the ability to directly manipulate gene expression through the production of transgenic or mutant chicken embryos by viral delivery methods has been useful to analyse gene function in a wide range of tissues, including the developing gonads. Chickens are amniotes and their development closely resembles that of mammals, implying underlying genetic conservation of key pathways, including sex development. Studies of sex determination and gonadal development in this model are providing insight into avian ovarian and testis developmental pathways and their evolution. Indeed, the chicken embryo is a suitable model for the functional analysis of genes implicated in human disorders of sex development, and studies in this model will complement those carried out in mammalian models such as the mouse. In this review we discuss the current knowledge of sex determination and sexual differentiation in avians, using chicken as model. We review how sex chromosomes contribute to this process and provide current information on our understanding of gonadal sexual differentiation at both the cellular and molecular level in the chicken embryo. Finally, we review the methods currently used to investigate the role of genes and signaling pathways during sexual differentiation, and discuss how these methods may contribute to further understanding of vertebrate gonadogenesis.

Transgenic chickens

The development of transgenic chicken technology has lagged far behind that of mammalian species. Two reasons for this are that only a one-cell-stage oocyte can be obtained from a sacrificed hen and that the yolk prevents high-magnification microscopic observation of oocytes. Recently, several new methods have been developed that will enable the successful establishment of transgenic chickens. Retroviral vectors are used in many cases because of their ability to incorporate transgenes into host cell chromosomes in a highly efficient manner. These viral vectors are injected directly into the embryos, usually at the blastodermal stage. In some cases, primordial germ cells (PGCs) are infected in vitro and then implanted into recipient embryos. Methods that do not rely on retroviral vectors are also available for creating transgenic chickens. Long-term culture of PGCs permits the selection of stably transfected cells and implantation of the manipulated PGCs. In addition, embryonic stem (ES) cell systems are available; however, the induction of functional gametes from ES cells has not, to our knowledge, been successful. It is clear that recent developments suggest that chickens may be used as a valuable experimental genetic system.

336 QUANTITATIVE ANALYSIS OF TETRACYCLINE-INDUCIBLE EXPRESSION OF THE GREEN FLUORESCENT PROTEIN GENE IN TRANSGENIC CHICKENS

The use of transgenic farm animals as bioreactors to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognised limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet (Kwon et al. 2011 Biochem. Biophys. Res. Commun. 410, 890–894). As a proof of principle study, however, quantitative assessment of expression was not possible, as only 1 G0 and 1 G1 transgenic chicken was obtained. In the current study, with 7 G2 transgenic chickens obtained from 1 G1 hen, we confirmed stable genomic integration of a single copy number of the transgene by Southern blot analysis. As we have observed in G1 transgenic chicken previously, all of the G2 transgenic chickens emitted a green fluorescence upon doxycycline feeding (50 mg kg–1 of formula feed). Fluorescence became detectable 4 days after starting doxycycline feeding, and maximum GFP expression was detected after 2 weeks. Removal of doxycycline from the diet after 14 days of induction feeding resulted in the return of external fluorescence to pre-induction levels after 39 days. Quantitative analysis of gene induction was done using protein and mRNA extracted from primary cultured cells derived from 6-day transgenic chicken embryos. The eggs were obtained by mating a nontransgenic wild-type hen with 1 of G2 transgenic roosters. Protein levels of GFP were analysed by immunoblot and quantified using a densitometer. In the absence of doxycycline, the amount of GFP per 1 µg of total protein was 0.2 ng. However, when the cells were treated with doxycycline for 6 days, the amount of GFP increased to 3.1 ng per 1 µg of total protein, which was 16-fold higher than that of the cells pre-treated with doxycycline. Switching to doxycycline-free medium after doxycycline induction resulted in significant abrogation of GFP expression in 6 days; the amount of GFP reduced from 3.1 to 0.5 ng, a 6.2-fold reduction. Transcription of the GFP gene was also assessed by Northern blot. The amount of GFP mRNA measured by band density increased as much as 20-fold (3.9/0.2) with 6 days of doxycycline induction and declined to 1/8 (3.9/0.5) when doxycycline was removed from the cell culture media for 6 days. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene.

DISEASE RESISTANCE AND OTHER APPLICATIONS OF TRANSGENESIS IN THE CHICKEN

Genetic modification of the chicken in terms of gene addition is now robust and efficient. Transgenes can be introduced by injection of lentiviral vectors into chick embryos or by transfection of transposon vectors into embryos or primordial germ cells in vitro. Lentiviral vectors are limited in the size of transgene they can incorporate but we have generated several different transgenic lines using HIV-derived vectors and have observed high levels of transgene expression and tissue-specific expression using regulatory sequences from several genes. M. McGrew (The Roslin Institute) has established primordial germ cell lines and effective methods for transfection with piggyBac and Tol2 transposon vectors. The primordial cells are injected into chick embryos where they populate the developing gonads and contribute to the germline in mature birds. The availability of primordial germ cell lines will also form the basis of using artificial site-specific nucleases for gene knockout and potentially gene targeting in the chicken. These technologies facilitate the application of transgenesis in the chicken for basic research and for potential applications in poultry breeding. The chick embryo is an invaluable model for studying vertebrate development as the embryos can be accessed in ovo or in culture at the earlier stages of development. Embryos can be transfected with transgenes by electroporation and manipulated to study many aspects of development. We are developing transgenic chickens in which fluorescent protein reporters are expressed either ubiquitously or in targeted cell types. These form the basis of novel tools for increasing the value of the chick embryo in studying development. We provide fertile eggs from these lines to other research groups and are investigating the development of macrophages using a macrophage-targeted reporter. The potential for the use of genetic modification to be used in poultry breeding can now be explored. Commercial poultry production is challenged by several major pathogens including avian influenza. Flocks can be protected by good biosecurity measures and/or vaccination but vaccination is not always effective. It may be possible to add novel genes to the chicken genome targeting avian influenza virus replication. We are developing this approach (with L. Tiley, Cambridge University) and have generated transgenic chickens that do not transmit avian influenza when directly infected with H5N1 virus.

형질전환 닭에서 GFP 유전자 전이 연구

Catholic University of Daegu School Medicine, 705-718, KoreaABSTRACT This study was performed to analyze the generational transmission and the expression of the foreign gene in the GFP transgenic chickens. The transmission rate and the expression of the GFP gene was investigated from the GFP transgenic rooster (G2) as the first founder to the ninth (G8). Analysis of GFP expression in hatched chickens was used the UV lamp. When GFP was expressed in the wings, bill and legs of a chick, the bird only was selected as a transgenic chick. The average transmission rate of the overall transgenic was 3858%. These results showed that the transmission of the GFP gene in the transgenic chickens in accordance with the laws of Mendel's continues to the next generation without gene silencing.(Key words : chicken, GFP, gene transmission)

Efficient production of transgenic chickens based on piggyBac

Transgenic techniques in chickens have been developed much more slowly than in mammals due to chickens' unique reproduction mechanism. Retroviral methods have been the most successful. piggyBac (PB) is a transposon that has a 13bp perfect terminal invert repeat sequence. PB can be inserted into TTAA sites and can also be precisely excised in mammals. Therefore, we have selected PB as a candidate to establish a new method to produce transgenic chickens. We constructed three donor vectors (ZGl-neo, ZGm-neo and ZGs-neo) expressing a GFP marker-gene and a neomycin resistant gene based on PB. We co-transfected each donor vector with a helper vector (CAG-PBase). We found that ZGl-neo was the most efficient PB vector. This vector could insert into TTAA sites in DF-1 cells. PB vectors were microinjected into sub-germinal cavity of newly laid eggs, and electroporation was then performed with a 20-V pulse for 5 cycles of 50ms on and 100ms off. GFP was expressed in different tissues of the embryos, including the gonads. Twenty-two chickens hatched after microinjection with compounds ZGl-neo and CAG-PBase (3:1). When we screened the blood DNA, 73% (16/22) of the individuals were positive. Thirteen of the chickens grew to adulthood, 11 of which were males. 40% (4/10) of the individuals were semen positive, and their copy numbers ranged from 0.05 to 0.21 (0.11, 0.21, 0.05, 0.06). No G1 offspring containing the integrated transposon were produced. We conclude that the PB transposon system is a novel useful tool for the efficient production of transgenic chickens.

Cholaminchloride hydrochloride-cationized gelatin/calcium-phosphate nanoparticles as gene carriers for transgenic chicken production

Currently, transgenic chickens are a popular method for pharmaceutical protein production. In this study, we employed cationized gelatin/calcium phosphate nanoparticles (ca-GCaPs) with surface modification by cholaminchloride hydrochloride as gene carriers to produce transgenic chickens. To evaluate the transfection efficiency, a plasmid (pEGFP-C1) was encapsulated in the ca-GCaPs for HeLa cells transfection. The ca-GCaPs were applied as gene carriers by direct injection into the area opaca of the chicken embryo blastodisc to produce the transgenic chicken. The particle size of the ca-GCaPs was 350nm and the zeta potential was +15mV. Plasmids encapsulated in the ca-GCaPs prevented from DNase I degradation. Based on biocompatibility analysis, the ca-GCaPs produce higher cell viability and less cytotoxicity compared to the commercially available product Lipofectamine™ 2000. The in vitro study indicated that the transfection rate using ca-GCaPs was higher than using cationized gelatin nanoparticles (ca-GPs) because calcium phosphate can provide osmotic pressure to break down lysosomes. In the in vivo study, the egg hatch rate was 100% after ca-GCaPs transfection, and green fluorescence protein expression could be observed in chicken tissue on the fourth day after transfection.

General steps in the production and establishment of transgenic chicken lines carrying a bacterial lacZ gene expressing β-galactosidase

Replication-defective retroviral vectors are efficient vehicles for the delivery of exogenous genes, and they may be used in the generation of transgenic animals. The replication-defective retroviral SNTZ vector carrying the lacZ gene with a nuclear localized signal was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the eggs were allowed to hatch, and the chickens were screened for the lacZ gene by using the polymerase chain reaction. Eight of 15 male chickens that survived to sexual maturity contained the lacZ gene in their semen. Subsequently, these males were mated with wild-type female chickens. In this study, performance of the SNTZ vector carrying the Eschrichia coli lacZ gene to express a nuclear-localized β-galactosidase was injected into the subgerminal cavity of embryos collected from newly laid eggs was reviewed.

PiggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens

Transgenic birds embody one of the most potent and exciting research tools in biotechnology for agriculture, medicine, and model animals. To date, retrovirus- or lentivirus-mediated transgenesis has been established in chickens and quail. However, despite having a valid technique for viral transduction to achieve transgenic birds, many obstacles exist for practical applications because of relatively low and variable rates of germ-line transmission and transgenic offspring showing transgene silencing, as well as safety issues related to viral vector use. Thus, the generation of transgenic poultry by nonviral integration is a prerequisite for the introduction of biotechnology to practical applications. Herein, we show that a germ-line-competent chicken primordial germ-cell (PGC) line was established with high efficiency of transmission to offspring and that piggyBac transposition into PGCs improved the efficiency of transgenic chicken production and led to high-level transgene expression. GFP transgene-expressing donor PGC-transferred recipient chickens produced donor-derived progenies, and the germ-line transmission efficiency of donor PGCs was 95.2% on average. Subsequently, half of the donor-derived offspring (52.2%) were transgenic chicks because GFP-expressing donor PGCs, in which a transgene was inserted into one chromosome 20, were heterozygous. In all of the transgenic chickens, GFP expression was constant and strong, regardless of age. Our results demonstrate that piggyBac transposition into the chicken PGC line could be the surest way to generate transgenic chickens safely for practical applications.

Overview on the efforts to generate transgenic chicken

After 25 years, the search for the avian cell that can be cultured indefinitely, genetically modified, and clonally derived while retaining its ability to enter the germ line has ended. Van de Lavoir et al. have defined the conditions for culture and genetic modification of primordial germ cells (PGCs) and showed that these cells are transmitted at high rates through the germline. The advent of this technology provides the ability to introduce transgenes of any size and to make site-specific changes to the genome. In this review, general avian reproduction and establishment of germ cells are described for possible routes to gene transfer. Also, the current gene transfer methodologies were summarized with a focus on PGC gene transfer. Finally, the application of transgenics to poultry was considered. Key words: Transgenic chickens, bioreactors, PGC.

Primordial germ cells (PGCs) as a tool for creating transgenic chickens

The transgenic chicken has great potential as a bioreactor for the production of valuable pharmaceutical proteins, notably in the oviduct/egg. Whereas conventional transgenic approaches have significant limitations in this species, an alternative approach employing primordial germ cells (PGCs), the progenitor cells to ova and spermatozoa, has now been successfully applied to the insertion of exogenous genes into birds. Recent developments in manipulating avian embryos make it possible to produce germline chimeras derived from transferred PGCs. In this review we describe the migration pathway of chicken PGCs during early development. We then summarize different methods for the isolation of PGCs and the diversity of techniques used to introduce genes into these cells. Finally, we describe an in vitro assay for testing tissue-specific vectors designed to express heterologous proteins in transgenic chickens.

Gelatin nanoparticles as gene carriers for transgenic chicken applications

To develop a safe and effective nonviral gene delivery system for transgenic chicken manipulation, we developed gelatin nanocarriers using a reporter plasmid (pEGFP-C1; enhanced green fluorescence protein, EGFP) that expressed EGFP. pEGFP-C1-containing gelatin nanoparticles (GP/pEGFP) were prepared using a water-ethanol solvent displacement method and characterized by size, surface charge, DNA loading, and DNA protection ability. For gene delivery, pEGFP-C1 was stably and efficiently encapsulated in GPs that were approximately 300 nm in diameter with a slight negative surface charge, which was prepared from gelatin solution at pH 8.0. Approximately, 85% of the plasmid DNA was encapsulated in the GPs. Electrophoresis results showed that the GPs provided protection against DNase I digestion. We used the GP/pEGFP as a vector to transfect cells and chicken embryos. The vector was nontoxic to cells, and GFP expression was effectively expressed 24 h after HeLa cell transfection. Direct injection was adapted for vector transport to the chicken embryo; injection in the area opaca (Ao) of the egg resulted in the highest hatching rate without affecting embryo development. GFP gene expression in embryo sections was observed 4 days after injection. The results of this study demonstrate that GPs are a suitable nonviral vector for delivering exogenous genes for transgenic chicken manipulation.

Quantitative Analysis of Tetracycline-Inducible Expression of the Green Fluorescent Protein Gene in Transgenic Chickens

The use of transgenic farm animals as "bioreactors" to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side-effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognized limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet. As a proof of principle study, however, quantitative assessment of expression was not possible, as only one G0 and one G1 transgenic chicken was obtained. In the current study, a sufficient number of G2 and G3 transgenic chickens were obtained, and quantification analysis demonstrated up to a 20-fold induction of expression by doxycycline. In addition, stable transmission of the transgene without any apparent genetic modifications was observed through several generations. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene. Importantly, these results also support the use of the retroviral system for generating transgenic animals with minimal risk in terms of biosafety.

Establishing Germline Chimeric Chickens Using Primordial Germ Cells

Chicken primordial germ cells (PGCs) are used to restore and conserve genetic resources of some endangered birds or flocks of domestic chickens. When PGCs are transferred from a donor to a recipient, they can survive and gain sexual maturity in the recipient chicken. This indicates that PGCs retaining exogenous genes can proliferate and develop in recipient gonads, and proves that transferred PGCs are effective sources for establishing transgenic and germline chimeric chickens. The PGC transfer method has been used to produce germline chimeric chickens and may become popular in the field of reproductive science. Germline chimeric chickens have the potential to reconstruct and reproduce endangered birds, including both domestic and wild species.

Lack of organ specific commitment of vagal neural crest cell derivatives as shown by back-transplantation of GFP chicken tissues

Neural crest cells (NCC) are multipotent progenitors that migrate extensively throughout the developing embryo and generate a diverse range of cell types. Vagal NCC migrate from the hindbrain into the foregut and from there along the gastrointestinal tract to form the enteric nervous system (ENS), the intrinsic innervation of the gut, and into the developing lung buds to form the intrinsic innervation of the lungs. The aim of this study was to determine the developmental potential of vagal NCC that had already colonised the gut or the lungs. We used transgenic chicken embryos that ubiquitously express green fluorescent protein (GFP) to permanently mark and fate-map vagal NCC using intraspecies grafting. This was combined with back-transplantation of gut and lung segments, containing GFP-positive NCC, into the vagal region of a second recipient embryo to determine, using immunohistochemical staining, whether gut or lung NCC are competent of re-colonising both these organs, or whether their fate is restricted. Chick(GFP)-chick intraspecies grafting efficiently labelled NCC within the gut and lung of chick embryos. When segments of embryonic day (E)5.5 pre-umbilical midgut containing GFP-positive NCC were back-transplanted into the vagal region of E1.5 host embryos, the GFP-positive NCC remigrated to colonise both the gut and lungs and differentiated into neurons in stereotypical locations. However, GFP-positive lung NCC did not remigrate when back-transplanted. Our studies suggest that gut NCC are not restricted to colonising only this organ, since upon back-transplantation GFP-positive gut NCC colonised both the gut and the lung.