Sang Su Shin

Generation of transgenic quail through germ cell-mediated germline transmission

Here, we describe the production of transgenic quail via a germline transmission system using postmigratory gonadal primordial germ cells (gPGCs). gPGCs retrieved from the embryonic gonads of 5‐day‐old birds were transduced with a lentiviral vector and subsequently transferred into recipient embryos. Testcross and genetic analyses revealed that among three germline chimeric G0 quail, one male produced transgenic offspring;of 310 hatchlings from the transgenic germline chimera, 24 were identified as donor‐derived offspring, and 6 were transgenic (6/310, 1.9%). Conventional transgenesis using stage × blastodermal embryos was also conducted, but the efficiency of transgenesis was similar between the two systems (<1.6 vs. 1.9% for the conventional and gPGC‐mediated systems, respectively). However, substantial advantages can be gained from gPGC‐mediated method in that it enables an induced germline modification, whereas direct retroviral transfer to stage X embryos causes mosaic integration. The use of gonadal PGCs for transgenesis may lead to the production of bioreactors.—Shin, S. S., Kim, T. M., Kim, S. Y., Kim, T. W., Seo, H. W., Lee, S. K., Kwon, S. C., Lee, G. S., Kim, H., Lim, J. M., Han, J. Y. Generation of transgenic quail through germ cell‐mediated germline transmission. FASEB J. 22, 2435–2444 (2008)

A Testis-Mediated Germline Chimera Production Based on Transfer of Chicken Testicular Cells Directly into Heterologous Testes

Abstract In this study, we proposed a testis-mediated germline chimera production system based on the transplantation of testicular cells directly into heterologous testes. The testicular cells of juvenile (4-wk-old) or adult (24-wk-old) Korean Ogol chickens with a recessive pigmentation inhibitory gene, with or without prior culture, were injected (2 × 107 cells/head) into the seminiferous tubules of juvenile or adult recipients with White Leghorn with a dominant pigmentation inhibitory gene in a 2 × 2 factorial arrangement. The localization of transplanted cells into the inner space of the seminiferous tubules was confirmed within 24 h after injection. Subsequent testcross analyses showed that 7.8% (5/64) of the recipients had chimeric status in their testes. The periods of time from transfer to hatching of the first progeny with black feathers were 38 and 45 days for adult cells transplanted into an adult recipient, 188 days for adult cells into a juvenile recipient, and 137 days for juvenile cells into a juvenile recipient. Culture of the testicular cells derived both colony-forming and monolayer-forming cells. The colony-forming cells were stained positively for periodic acid Schiff solution, and further reacted with anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibodies both before and after culture for 15 days. In conclusion, it may be possible to develop the testis-mediated germline chimera production technique, which extends the feasibility of genetic manipulations in avian species.

Production of Biofunctional Recombinant Human Interleukin 1 Receptor Antagonist (rhIL1RN) from Transgenic Quail Egg White

Oviduct-specific expression of heterologous recombinant proteins in transgenic birds is a promising technology for the large-scale production of therapeutic proteins in eggs. We describe the production of recombinant human interleukin 1 receptor antagonist (rhIL1RN) in the eggs of transgenic quails. To drive tissue-specific expression of rhIL1RN, a 1.35-kb fragment of the chicken ovalbumin promoter, which contains both the steroid-dependent regulatory element and the negative regulatory element, was used. A transgenic quail was generated by microinjection of a concentrated stock of lentivirus into stage X blastodermal cells. A single copy of the transgene was integrated into the seventh intron of the gene for conserved oligomeric golgi complex protein 5 (COG5) on chromosome 1. As expected, rhIL1RN expression was restricted to oviductal tissue, and the amount of protein deposited in the eggs of homozygous transgenic quails ranged from 88.7 to 233.8 ng/ml. Transgene expression was conserved from the G1 generation to the G4 generation, and there was no evidence of transgene silencing. In a bioassay using the EL4.NOB-1/CTLL-2 coculture system, no significant difference was observed between the egg-produced rhIL1RN and a commercially available rhIL1RN (anakinra).