Transgenesis and genome editing in birds are based on a unique germline transmission system using primordial germ cells (PGCs), which is quite different from the mammalian transgenic and genome editing system. PGCs are progenitor cells of gametes that can deliver genetic information to the next generation. Since avian PGCs were first discovered in nineteenth century, there have been numerous efforts to reveal their origin, specification, and unique migration pattern, and to improve germline transmission efficiency. Recent advances in the isolation and in vitro culture of avian PGCs with genetic manipulation and genome editing tools enable the development of valuable avian models that were unavailable before. However, many challenges remain in the production of transgenic and genome-edited birds, including the precise control of germline transmission, introduction of exogenous genes, and genome editing in PGCs. Therefore, establishing reliable germline-competent PGCs and applying precise genome editing systems are critical current issues in the production of avian models. Here, we introduce a historical overview of avian PGCs and their application, including improved techniques and methodologies in the production of transgenic and genome-edited birds, and we discuss the future potential applications of transgenic and genome-edited birds to provide opportunities and benefits for humans.

/pdf/primordial-germ-cell-mediated-transgenesis-and-genome-50g8pnzssy.pdf

Primordial germ cell-mediated transgenesis and genome editing in birds.

This review provides an historic perspective of the key steps from those reported at the 1st Transgenic Animal Research Conference in 1997 through to the very latest developments in avian transgenesis. Eighteen years later, on the occasion of the 10th conference in this series, we have seen breakthrough advances in the use of viral vectors and transposons to transform the germline via the direct manipulation of the chicken embryo, through to the establishment of PGC cultures allowing in vitro modification, expansion into populations to analyse the genetic modifications and then injection of these cells into embryos to create germline chimeras. We have now reached an unprecedented time in the history of chicken transgenic research where we have the technology to introduce precise, targeted modifications into the chicken genome, ranging from; new transgenes that provide improved phenotypes such as increased resilience to economically important diseases; the targeted disruption of immunoglobulin genes and replacement with human sequences to generate transgenic chickens that express "humanised" antibodies for biopharming; and the deletion of specific nucleotides to generate targeted gene knockout chickens for functional genomics. The impact of these advances is set to be realised through applications in chickens, and other bird species as models in scientific research, for novel biotechnology and to protect and improve agricultural productivity.

Advances in genetic engineering of the avian genome: "Realising the promise".

The role of PPAR alpha in perfluorooctanoic acid induced developmental cardiotoxicity and l-carnitine mediated protection–Results of in ovo gene silencing

Due to its amenability to manipulations, to live observation and its striking similarities to mammals, the chicken embryo has been one of the major animal models in biomedical research. Although it is technically possible to genome-edit the chicken, its long generation time (6 months to sexual maturity) makes it an impractical lab model and has prevented it widespread use in research. The Japanese quail (Coturnix coturnix japonica) is an attractive alternative, very similar to the chicken, but with the decisive asset of a much shorter generation time (1.5 months). In recent years, transgenic quail lines have been described. Most of them were generated using replication-deficient lentiviruses, a technique that presents diverse limitations. Here, we introduce a novel technology to perform transgenesis in quail, based on the in vivo transfection of plasmids in circulating Primordial Germ Cells (PGCs). This technique is simple, efficient and allows using the infinite variety of genome engineering approaches developed in other models. Furthermore, we present a website centralizing quail genomic and technological information to facilitate the design of genome-editing strategies, showcase the past and future transgenic quail lines and foster collaborative work within the avian community.

Transgenesis and web resources in quail.

In this work we have determined molecular signatures of oviduct epithelial and progenitor cells. We have proposed a panel of selected marker genes, which correspond with the phenotype of oviduct cells of a laying hen (Gallus gallus domesticus) and quail (Coturnix japonica). We demonstrated differences in characteristics of those cells, in tissue and in vitro, with respect to different anatomical and functional parts of the oviduct (infundibulum (INF), distal magnum (DM, and proximal magnum (PM)). The following gene expression signatures were studied: (1) oviduct markers (estrogen receptor 1, ovalbumin, and SPINK7 - ovomucoid), (2) epithelial markers (keratin 5, keratin 14, and occludin) and (3) stem-like/progenitor markers (CD44 glycoprotein, LGR5, Musashi-1, and sex determining region Y-box 9, Nanog homebox, OCT4/cPOUV gene encoding transcription factor POU5F3). In chicken, the expression of oviduct markers increased toward the proximal oviduct. Epithelial markers keratin14 and occludin were high in distal oviduct and decreased toward the proximal magnum. In quail oviduct tissue, the gene expression pattern of oviduct/epithelial markers was similar to chicken. The markers of progenitors/stemness in hen oviduct (Musashi-1 and CD44 glycoprotein) had the highest relative expression in the infundibulum and decreased toward the proximal magnum. In quail, we found significant expression of four progenitor markers (LGR5 gene, SRY sex determining region Y-box 9, OCT4/cPOUV gene, and CD44 glycoprotein) that were largely present in the distal oviduct. After in vitro culture of oviduct cells, the gene expression pattern has changed. High secretive potential of magnum-derived cells diminished by using decreased abundance of mRNA. On the other hand, chicken oviduct cells originating from the infundibulum gained ability to express OVM and OVAL. Epithelial character of the cells was maintained in vitro. Among progenitor markers, both hen and quail cells expressed high level of SOX9, LGR5 and Musashi-1. Analysis of tissue material revealed gradual increase/decrease pattern in majority of the oviduct markers in both species. This pattern changed after the oviductal cells have been cultured in vitro. The results can provide molecular tools to validate the phenotype of in vitro biological models from reproductive tissue.

/pdf/molecular-signatures-of-epithelial-oviduct-cells-of-a-laying-1v6at6lt5n.pdf

Molecular signatures of epithelial oviduct cells of a laying hen (Gallus gallus domesticus) and quail (Coturnix japonica).

Several strategies have been used to generate transgenic birds. The most successful method so far has been the injection of lentiviral vectors into the subgerminal cavity of a newly laid egg. We report here a new, easy and effective way to produce transgenic quails through direct injection of a lentiviral vector, containing an enhanced-green fluorescent protein (eGFP) transgene, into the blood vessels of quail embryos at Hamburger-Hamilton stage 13–15 (HH13–15). A total of 80 embryos were injected and 48 G0 chimeras (60%) were hatched. Most injected embryo organs and tissues of hatched quails were positive for eGFP. In five out of 21 mature G0 male quails, the semen was eGFP-positive, as detected by polymerase chain reaction (PCR), indicating transgenic germ line chimeras. Testcross and genetic analyses revealed that the G0 quail produced transgenic G1 offspring; of 46 G1 hatchlings, 6 were transgenic (6/46, 13.0%). We also compared this new method with the conventional transgenesis using stage X subgerminal cavity injection. Total 240 quail embryos were injected by subgerminal cavity injection, of which 34 (14.1%) were hatched, significantly lower than the new method. From these hatched quails semen samples were collected from 19 sexually matured males and tested for the transgene by PCR. The transgene was present in three G0 male quails and only 4/236 G1 offspring (1.7%) were transgenic. In conclusion, we developed a novel bird transgenic method by injection of lentiviral vector into embryonic blood vessel at HH 13–15 stage, which result in significant higher transgenic efficiency than the conventional subgerminal cavity injection.

/pdf/transgenic-quail-production-by-microinjection-of-lentiviral-13pj8cw12i.pdf

Transgenic quail production by microinjection of lentiviral vector into the early embryo blood vessels.

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Transgenic quail production by microinjection of lentiviral vector into the early embryo blood vessels.