title: Generation of cloned mice from adult neurons by direct nuclear transfer.
abstract: Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain "unclonable" for unknown reasons. Here, using a combination of two epigenetic approaches, we examined whether neurons from adult mice could be cloned. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark-dimethylated histone H3 lysine 9 (H3K9me2)-and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (of embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrates, to our knowledge for the first time, that adult neurons can be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos.
authors: RIKEN BioResource Center, Tsukuba, Ibaraki, Japan Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi, Japan.
authors: Eiji
authors: E
authors: Mizutani
authors: RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
authors: Mami
authors: M
authors: Oikawa
authors: Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors: Hidetoshi
authors: H
authors: Kassai
authors: RIKEN BioResource Center, Tsukuba, Ibaraki, Japan Graduate School of Life and Environmental Science, University of Tsukuba, Ibaraki, Japan.
authors: Kimiko
authors: K
authors: Inoue
authors: RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
authors: Hirosuke
authors: H
authors: Shiura
authors: RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
authors: Ryutaro
authors: R
authors: Hirasawa
authors: RIKEN BioResource Center, Tsukuba, Ibaraki, Japan Graduate School of Life and Environmental Science, University of Tsukuba, Ibaraki, Japan.
authors: Satoshi
authors: S
authors: Kamimura
authors: RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
authors: Shogo
authors: S
authors: Matoba
authors: RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
authors: Narumi
authors: N
authors: Ogonuki
authors: Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi, Japan.
authors: Hiroaki
authors: H
authors: Nagatomo
authors: RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
authors: Kuniya
authors: K
authors: Abe
authors: Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi, Japan.
authors: Teruhiko
authors: T
authors: Wakayama
authors: Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors: Atsu
authors: A
authors: Aiba
authors: RIKEN BioResource Center, Tsukuba, Ibaraki, Japan Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Graduate School of Life and Environmental Science, University of Tsukuba, Ibaraki, Japan ogura@rtc.riken.go.jp.
authors: Atsuo
authors: A
authors: Ogura
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D047108:Embryonic Development; D005260:Female; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D056466:Histone Demethylases / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D023421:Models, Animal; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D011689:Purkinje Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
chemical_list: D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C012589:trichostatin A; D056466:Histone Demethylases
keywords: CA1 pyramidal cells; assisted reproductive technology; cloning; developmental biology; early development; hippocampus; mouse; neural cells; nuclear transfer; reprogramming