title: The meganuclease I-SceI containing nuclear localization signal (NLS-I-SceI) efficiently mediated mammalian germline transgenesis via embryo cytoplasmic microinjection.
abstract: The meganuclease I-SceI has been effectively used to facilitate transgenesis in fish eggs for nearly a decade. I-SceI-mediated transgenesis is simply via embryo cytoplasmic microinjection and only involves plasmid vectors containing I-SceI recognition sequences, therefore regarding the transgenesis process and application of resulted transgenic organisms, I-SceI-mediated transgenesis is of minimal bio-safety concerns. However, currently no transgenic mammals derived from I-SceI-mediated transgenesis have been reported. In this work, we found that the native I-SceI molecule was not capable of facilitating transgenesis in mammalian embryos via cytoplasmic microinjection as it did in fish eggs. In contrast, the I-SceI molecule containing mammalian nuclear localization signal (NLS-I-SceI) was shown to be capable of transferring DNA fragments from cytoplasm into nuclear in porcine embryos, and cytoplasmic microinjection with NLS-I-SceI mRNA and circular I-SceI recognition sequence-containing transgene plasmids resulted in transgene expression in both mouse and porcine embryos. Besides, transfer of the cytoplasmically microinjected mouse and porcine embryos into synchronized recipient females both efficiently resulted in transgenic founders with germline transmission competence. These results provided a novel method to facilitate mammalian transgenesis using I-SceI, and using the NLS-I-SceI molecule, a simple, efficient and species-neutral transgenesis technology based on embryo cytoplasmic microinjection with minimal bio-safety concerns can be established for mammalian species. As far as we know, this is the first report for transgenic mammals derived from I-SceI-mediated transgenesis via embryo cytoplasmic microinjection.
authors.affiliation: Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China.
authors.forename: Xiao-Yang
authors.initials: XY
authors.lastname: Zhou
authors.affiliation: Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China.
authors.forename: Peng-Ying
authors.initials: PY
authors.lastname: Xiang
authors.affiliation: Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China.
authors.forename: Lu-Lu
authors.initials: LL
authors.lastname: Wang
authors.affiliation: China Three Gorges Museum, Chongqing, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Tang
authors.affiliation: Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Xie
authors.affiliation: Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Wei
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D003593:Cytoplasm / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian; D005260:Female; D018014:Gene Transfer Techniques / Q000662:veterinary*; D051379:Mice / Q000196:embryology / Q000235:genetics; D008845:Microinjections / Q000379:methods*; D019913:Nuclear Localization Signals / Q000235:genetics* / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism*; D013552:Swine; D013556:Swine, Miniature / Q000196:embryology / Q000235:genetics
chemical_list: D019913:Nuclear Localization Signals; D015252:Deoxyribonucleases, Type II Site-Specific