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      PLoS ONE
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          Abstract

          Microcell-mediated chromosome transfer (MMCT) is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs) as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses. Using this method, we transferred MACs to NIH3T3 cells with 26.5 times greater efficiency than that obtained using the conventional MMCT method. Retro-MMCT was applicable to a variety of recipient cells, including embryonic stem cells. Moreover, retro-MMCT enabled efficient transfer of MAC to recipient cells derived from humans, monkeys, mice, rats, and rabbits. These results demonstrate the utility of retro-MMCT for the efficient transfer of chromosomes to various types of target cell.

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          Most cited references25

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          Complete Genetic Correction of iPS Cells From Duchenne Muscular Dystrophy

          Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Induced pluripotent stem (iPS) cells have great potential for gene therapy, as such cells can be generated from the individual's own tissues, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we show herein the complete correction of a genetic deficiency in iPS cells derived from Duchenne muscular dystrophy (DMD) model (mdx) mice and a human DMD patient using a HAC with a complete genomic dystrophin sequence (DYS-HAC). Deletion or mutation of dystrophin in iPS cells was corrected by transferring the DYS-HAC via microcell-mediated chromosome transfer (MMCT). DMD patient- and mdx-specific iPS cells with the DYS-HAC gave rise to differentiation of three germ layers in the teratoma, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice from mdx-iPS (DYS-HAC) cells were produced and DYS-HAC was detected in all tissues examined, with tissue-specific expression of dystrophin. Therefore, the combination of patient-specific iPS cells and HAC-containing defective genes represents a powerful tool for gene and cell therapies.
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            Cloned transchromosomic calves producing human immunoglobulin.

            Human polyclonal antibodies (hPABs) are useful therapeutics, but because they are available only from human donors, their supply and application is limited. To address this need, we prepared a human artificial chromosome (HAC) vector containing the entire unrearranged sequences of the human immunoglobulin (hIg) heavy-chain (H) and lambda (lambda) light-chain loci. The HAC vector was introduced into bovine primary fetal fibroblasts using a microcell-mediated chromosome transfer (MMCT) approach. Primary selection was carried out, and the cells were used to produce cloned bovine fetuses. Secondary selection was done on the regenerated fetal cell lines, which were then used to produce four healthy transchromosomic (Tc) calves. The HAC was retained at a high rate (78-100% of cells) in calves and the hIg loci underwent rearrangement and expressed diversified transcripts. Human immunoglobulin proteins were detected in the blood of newborn calves. The production of Tc calves is an important step in the development of a system for producing therapeutic hPABs.
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              Functional expression and germline transmission of a human chromosome fragment in chimaeric mice.

              Human chromosomes or chromosome fragments derived from normal fibroblasts were introduced into mouse embryonic stem (ES) cells via microcell-mediated chromosome transfer (MMCT) and viable chimaeric mice were produced from them. Transferred chromosomes were stably retained, and human genes, including immunoglobulin (Ig) kappa, heavy, lambda genes, were expressed in proper tissue-specific manner in adult chimaeric tissues. In the case of a human chromosome (hChr.) 2-derived fragment, it was found to be transmitted to the offspring through the germline. Our study demonstrates that MMCT allows for introduction of very large amounts of foreign genetic material into mice. This novel procedure will facilitate the functional analyses of human genomes in vivo.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                7 June 2016
                2016
                : 11
                : 6
                : e0157187
                Affiliations
                [1 ]Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
                [2 ]Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Tottori, Japan
                [3 ]Chromosome Engineering Research Center, Tottori University, Tottori, Japan
                Imperial College London, UNITED KINGDOM
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: TS. Performed the experiments: TS. Analyzed the data: TS TH. Contributed reagents/materials/analysis tools: YK MO. Wrote the paper: TS TH.

                Author information
                http://orcid.org/0000-0001-8162-8809
                Article
                PONE-D-16-12671
                10.1371/journal.pone.0157187
                4896634
                27271046
                1f4e0003-8d6e-45e4-83ca-15f74046ce4f
                © 2016 Suzuki et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 29 March 2016
                : 25 May 2016
                Page count
                Figures: 4, Tables: 1, Pages: 11
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001691, Japan Society for the Promotion of Science;
                Award ID: 24790696
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001691, Japan Society for the Promotion of Science;
                Award ID: 15K18392
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001691, Japan Society for the Promotion of Science;
                Award ID: 23390256
                Award Recipient :
                Funded by: Regional Innovation Strategy Support Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan
                Award Recipient :
                This work was supported in part by the Japan Society for the Promotion of Science KAKENHI Grant numbers 24790696, 15K18392 (T.S.) and 23390256 (T.H.) ( http://www.jsps.go.jp/english/index.html); Regional Innovation Strategy Support Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (M.O.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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