title: Establishment of medakafish as a model for stem cell-based gene therapy: efficient gene delivery and potential chromosomal integration by baculoviral vectors.
abstract: Viral vectors hold promise and challenges in gene therapy. Specifically, we have previously shown that baculoviral (BV) vectors have a high efficiency of gene delivery in human embryonic stem (ES) cells. Here we report the development of a complementary system to further our evaluation by utilizing the laboratory fish medaka that has ES cell lines and tools for experimental analyses in vitro and in vivo. We show that BV vectors can give rise to almost 100% of transient gene delivery in the medaka ES cell line MES1. BV-transduced MES1 cells reproducibly (at approximately 10(-5)) produce GFP-expressing colonies that, upon manual isolation, develop into stable clones during 300 days of culture. Surprisingly, BV transduction can also mediate efficient gene integration in the medaka genome, as fluorescent in situ hybridization revealed the presence of the BV-delivered gfp transgene in multiple locations in nuclei and on various chromosomes of metaphase spreads. We show that BV transduction does not compromise the genome stability and pluripotency of MES1 cells. We conclude that BV can efficiently mediate gene delivery and chromosomal integration in medaka ES cells. Therefore, medaka provides a powerful system for analyzing the potential of BV-mediated gene delivery in stem cells and gene therapy.
authors.affiliation: Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, 637457 Singapore.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yan
authors.forename: Juan
authors.initials: J
authors.lastname: Du
authors.forename: Tiansheng
authors.initials: T
authors.lastname: Chen
authors.forename: Meisheng
authors.initials: M
authors.lastname: Yi
authors.forename: Mingyou
authors.initials: M
authors.lastname: Li
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
authors.forename: Chang Ming
authors.initials: CM
authors.lastname: Li
authors.forename: Yunhan
authors.initials: Y
authors.lastname: Hong
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002678:Chimera / Q000033:anatomy & histology / Q000502:physiology; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D009990:Oryzias / Q000033:anatomy & histology / Q000196:embryology / Q000235:genetics*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology; D033581:Stem Cell Transplantation; D014018:Tissue Distribution; D019076:Transgenes