title: Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector, RCASBP.
abstract: Functional genomics in avian models has lagged behind that of mammals, and the production of transgenic birds has proven to be challenging and time-consuming. All current methods rely upon breeding chimeric birds through at least one generation. Here, we report a rapid method for the ubiquitous expression of GFP in chicken embryos in a single generation (G-0), using the avian retroviral vector, Replication-Competent Avian sarcoma-leukosis virus, with a Splice acceptor, Bryan RSV Pol (RCASBP). High-titre RCASBP retrovirus carrying eGFP was injected into unincubated (stage X) blastoderms in ovo. This resulted in stable and widespread expression of eGFP throughout development in a very high proportion of embryos. Transgenic tissues were identified by fluorescence and immunohistochemistry. These results indicate that chicken blastodermal cells are permissive for infection by the RCASBP virus. This system represents a rapid and efficient method of producing global gene expression in the chicken embryo. The method can be used to generate avian cells with a stable genetic marker, or to induce global expression of a gene of choice. Interestingly, in day 8.5 embryos, somatic cells the embryonic gonads were predominantly GFP positive but primordial germ cells were GFP negative, indicating viral silencing in the embryonic germline. This dichotomy in the gonads allows the isolation or enrichment of the germ cells through negative selection during embryonic stages. This transgenic chicken model is of value in developmental studies, and for the isolation and study of avian primordial germ cells.
authors.affiliation: Murdoch Childrens Research Institute, University of Melbourne, Department of Paediatrics, Royal Children's Hospital, Flemington Road, Parkville, Melbourne, Victoria 3052, Australia. craig.smith@mcri.edu.au
authors.forename: Craig A
authors.initials: CA
authors.lastname: Smith
authors.forename: Kelly N
authors.initials: KN
authors.lastname: Roeszler
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Sinclair
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001756:Blastoderm / Q000821:virology; D002642:Chick Embryo; D002645:Chickens / Q000235:genetics*; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*
chemical_list: D049452:Green Fluorescent Proteins