title: Generation of domestic transgenic cloned kittens using lentivirus vectors.
abstract: The efficient use of somatic cell nuclear transfer (SCNT), in conjunction with genetic modification of donor cells provides a general means to add or inactivate genes in mammals. This strategy has substantially improved the efficacy of producing genetically identical animals carrying mutant genes corresponding to specific human disorders. Lentiviral (LV) vectors have been shown to be well suited for introducing transgenes into cells to be used as donor nuclei for SCNT. In the present study, we established an LV vector-based transgene delivery approach for producing live transgenic domestic cats by SCNT. We have demonstrated that cat fetal fibroblasts can be transduced with EGFP-encoding LV vectors bearing various promoters including the human cytomegalovirus immediate early (hCMV-IE) promoter, the human translation elongation factor 1alpha (hEF-1alpha) promoter and the human ubiquitin C (hUbC) promoter. Among the promoters tested, embryos reconstructed with donor cells transduced with a LV-vector bearing the hUbC promoter displayed sustained transgene expression at the blastocyst stage while embryos reconstructed with LV vector-transduced cells containing hCMV-IE-EGFP or hEF-1alpha-EGFP cassettes did not. After transfer of 291 transgenic cloned embryos into the oviducts of eight recipient domestic cats (mean =36.5 +/- 10.1), three (37.5%) were diagnosed to be pregnant, and a total of six embryos (2.1%) implanted. One live male offspring was delivered by Cesarean section on day 64 of gestation, and two kittens were born dead after premature delivery on day 55. In summary, we report the birth of transgenic cloned kittens produced by LV vector-mediated transduction of donor cells and confirm that cloned kittens express the EGFP reporter transgene in all body tissues.
authors.affiliation: Audubon Center for Research of Endangered Species, New Orleans, Louisiana 70131, USA. mgomez@auduboninstitute.org
authors.forename: Martha C
authors.initials: MC
authors.lastname: Gómez
authors.forename: Charles Earle
authors.initials: CE
authors.lastname: Pope
authors.forename: Robert H
authors.initials: RH
authors.lastname: Kutner
authors.forename: David M
authors.initials: DM
authors.lastname: Ricks
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Lyons
authors.forename: Mark T
authors.initials: MT
authors.lastname: Ruhe
authors.forename: Cherie
authors.initials: C
authors.lastname: Dumas
authors.forename: Justine
authors.initials: J
authors.lastname: Lyons
authors.forename: Betsy L
authors.initials: BL
authors.lastname: Dresser
authors.forename: Jakob
authors.initials: J
authors.lastname: Reiser
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000502:physiology; D001755:Blastocyst / Q000502:physiology; D002415:Cats / Q000235:genetics*; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus; D008297:Male; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D011401:Promoter Regions, Genetic; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins