title: Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons.
abstract: The derivation of germ-line competent avian primordial germ cells establishes a cell-based model system for the investigation of germ cell differentiation and the production of genetically modified animals. Current methods to modify primordial germ cells using DNA or retroviral vectors are inefficient and prone to epigenetic silencing. Here, we validate the use of transposable elements for the genetic manipulation of primordial germ cells. We demonstrate that chicken primordial germ cells can be modified in vitro using transposable elements. Both piggyBac and Tol2 transposons efficiently transpose primordial germ cells. Tol2 transposon integration sites were spread throughout both the macro- and microchromosomes of the chicken genome and were more prevalent in gene transcriptional units and intronic regions, consistent with transposon integrations observed in other species. We determined that the presence of insulator elements was not required for reporter gene expression from the integrated transposon. We further demonstrate that a gene-trap cassette carried in the Tol2 transposon can trap and mutate endogenous transcripts in primordial germ cells. Finally, we observed that modified primordial germ cells form functional gametes as demonstrated by the generation of transgenic offspring that correctly expressed a reporter gene carried in the transposon. Transposable elements are therefore efficient vectors for the genetic manipulation of primordial germ cells and the chicken genome.
authors.affiliation: Division of Developmental Biology, The Roslin Institute and Royal Dick School of Veterinary Studies,  University of Edinburgh, Midlothian, EH25 9RG,  United Kingdom.
authors.forename: Joni
authors.initials: J
authors.lastname: Macdonald
authors.forename: Lorna
authors.initials: L
authors.lastname: Taylor
authors.forename: Adrian
authors.initials: A
authors.lastname: Sherman
authors.forename: Koichi
authors.initials: K
authors.lastname: Kawakami
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Helen M
authors.initials: HM
authors.lastname: Sang
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McGrew
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D015139:Blotting, Southern; D018929:Cell Culture Techniques; D002642:Chick Embryo; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D005854:Germ Cells / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D017931:DNA Primers; D004251:DNA Transposable Elements