Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer

BMC Biotechnology

Research Article

Yasuhiro Kazuki¹  Mitsuo Oshimura¹  Takafumi Nakamura²  Kanako Kazuki³  Naoyo Kajitani³  Masato Takiguchi³  Yuji Nakayama⁴  Motonobu Katoh⁵ 

[1] Chromosome Engineering Research Center, Tottori University, 683-8503, Yonago, Japan;Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 683-8503, Yonago, Japan;Core Research for Evolutional Science and Technology (CREST) project, Japan Science and Technology Agency, 332-0012, Kawaguchi, Japan;Core Facility for Therapeutic Vectors, The Institute of Medical Science, The University of Tokyo, 108-8639, Tokyo, Japan;RNA and Biofunctions, Precursory Research for Embryonic Science and Technology (PRESTO) project, Japan Science and Technology Agency, 332-0012, Kawaguchi, Japan;Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 683-8503, Yonago, Japan;Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, 683-8503, Yonago, Japan;Division of Human Genome Science, Department of Molecular and Cellular Biology, Faculty of Medicine, Tottori University, 683-8503, Yonago, Japan;Chromosome Engineering Research Center, Tottori University, 683-8503, Yonago, Japan;

关键词: HT1080 Cell;    Measle Virus;    Recipient Cell;    Syncytium Formation;    Human Artificial Chromosome;   

DOI  :  10.1186/1472-6750-10-37

 received in 2009-10-15, accepted in 2010-05-06,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundMicrocell-mediated chromosome transfer (MMCT) is a technique by which a chromosome(s) is moved from donor to recipient cells by microcell fusion. Polyethylene glycol (PEG) has conventionally been used as a fusogen, and has been very successful in various genetic studies. However, PEG is not applicable for all types of recipient cells, because of its cell type-dependent toxicity. The cytotoxicity of PEG limits the yield of microcell hybrids to low level (10-6 to 10-5 per recipient cells). To harness the full potential of MMCT, a less toxic and more efficient fusion protocol that can be easily manipulated needs to be developed.ResultsMicrocell donor CHO cells carrying a human artificial chromosome (HAC) were transfected with genes encoding hemagglutinin (H) and fusion (F) proteins of an attenuated Measles Virus (MV) Edmonston strain. Mixed culture of the CHO transfectants and MV infection-competent human fibrosarcoma cells (HT1080) formed multinucleated syncytia, suggesting the functional expression of the MV-H/F in the CHO cells. Microcells were prepared and applied to HT1080 cells, human immortalized mesenchymal stem cells (hiMSC), and primary fibroblasts. Drug-resistant cells appeared after selection in culture with Blasticidin targeted against the tagged selection marker gene on the HAC. The fusion efficiency was determined by counting the total number of stable clones obtained in each experiment. Retention of the HAC in the microcell hybrids was confirmed by FISH analyses. The three recipient cell lines displayed distinct fusion efficiencies that depended on the cell-surface expression level of CD46, which acts as a receptor for MV. In HT1080 and hiMSC, the maximum efficiency observed was 50 and 100 times greater than that using conventional PEG fusion, respectively. However, the low efficiency of PEG-induced fusion with HFL1 was not improved by the MV fusogen.ConclusionsEctopic expression of MV envelope proteins provides an efficient recipient cell-oriented MMCT protocol, facilitating extensive applications for studies of gene function and genetic corrections.

【 授权许可】

Unknown   
© Katoh et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.