title: Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss.
abstract: Cre/loxP-mediated DNA excision in germ cell lineages could contribute substantially to the study of germ cell biology in salmonids, which are emerging as a model species in this field. However, a cell type-specific Cre/loxPsystem has not been successfully developed for any salmonid species. Therefore, we examined the feasibility of Cre/loxP-mediated, germ cell-specific gene excision and transgene activation in rainbow trout. Double-transgenic (wTg) progeny were obtained by mating a transgenic male carryingcrewith a transgenic female carrying thehsc-LRLGgene;crewas driven by rainbow troutvasaregulatory regions and thehsc-LRLGgene was made up of the rainbow troutheat-shock-cognate71promoter, theDsRedgene flanked by twoloxPsites, and theEgfpgene. PCR analysis, fluorescence imaging, and histological analysis revealed that excision of theloxP-flanked sequence and activation ofEgfpoccurred only in germ cells of wTg fish. However, progeny tests revealed that the excision efficiency ofloxP-flanked sequence in germ cells was low (≤3.27%). In contrast, the other wTg fish derived from two differentcre-transgenic males frequently excised theloxP-flanked sequence in germ cells (≤89.25%). Thus, we showed for the first time successful germ cell-specific transgene manipulation via the Cre/loxPsystem in rainbow trout. We anticipate that this technology will be suitable for studies of cell function through cell targeting, cell-linage tracing, and generating cell type-specific conditional gene knockouts and separately for developing sterile rainbow trout in aquaculture.
authors.affiliation: Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan Research Fellow of the Japan Society for the Promotion of Science, Tokyo, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Katayama
authors.affiliation: Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan.
authors.forename: Sachi
authors.initials: S
authors.lastname: Kume
authors.affiliation: Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Hattori-Ihara
authors.affiliation: Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan.
authors.forename: Sakiko
authors.initials: S
authors.lastname: Sadaie
authors.affiliation: Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan Life Science Center of Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Ibaraki, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hayashi
authors.affiliation: Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan goro@kaiyodai.ac.jp.
authors.forename: Goro
authors.initials: G
authors.lastname: Yoshizaki
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D005854:Germ Cells; D019426:Integrases; D017686:Oncorhynchus mykiss / Q000235:genetics*; D019076:Transgenes
chemical_list: C045073:Cre recombinase; D019426:Integrases
keywords: Cre/loxP system; germ cell; rainbow trout; transgenic fish