Generation of Cloned Mice from Adult Neurons by Direct Nuclear Transfer1

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.

The Complete Genome Sequences, Unique Mutational Spectra, and Developmental Potency of Adult Neurons Revealed by Cloning

“…By generating neonatal and fertile adult mice from genetically labeled MT neurons derived from mice up to 4.5 months old, we demonstrate that despite the difficulty in inducing neurons to re-enter the cell cycle, at least some neurons maintain sufficiently plastic and intact genomes to produce an entire animal. While other studies have reported varying degrees of success (or failure) in efforts to clone from non-embryonic cortical neurons, this study is unique because we conclusively demonstrate the donor cell origin and age, and produce the first clones from adult mice older than 6-8 weeks of age (Kawase et al, 2000; Makino et al, 2005; Mizutani et al, 2015; Osada et al, 2002; Osada et al, 2005; Wakayama et al, 1998; Yamazaki et al, 2001). In addition, because we have deciphered full MT neuronal genome sequences, we show that accumulation of mutations during the development and post-mitotic aging of neurons is compatible with seemingly normal embryonic and postnatal development, which has relevance for the use of adult cells in regenerative medicine.…”

From cloned frogs to patient matched stem cells: induced pluripotency or somatic cell nuclear transfer?

“…In the absence of the HDAC inhibitor, a similar decline in re-cloning efficiency was observed (Figure 2d). These and numerous other studies reporting the cloning of animals from adult somatic cells, including from adult neurons [32 ], point to a generally greater ability of nuclear transfer to restore full developmental competence to a somatic nucleus than iPS reprogramming. But a systematic and direct comparison of the complementation potential of ntES cells and iPS cells from adult somatic cells is yet to be performed.…”

Development of reproductive engineering techniques at the RIKEN BioResource Center

“…This enables the researcher to identify the effects of the cell type and genotype on cloning efficiency. Following the first birth of cloned mice by Dr. Wakayama [ 83 ], we cloned 13 cell types for the production of cloned mice [ 50 , 66 ]. The conclusions drawn from our research are as follows.…”