Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer.
Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer.
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DOI：
10.1186/1472-6750-10-37
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发表时间：
2010-05-06
影响因子：
3.5
通讯作者：
Oshimura M
中科院分区：
文献类型：
--
作者：
Katoh M;Kazuki Y;Kazuki K;Kajitani N;Takiguchi M;Nakayama Y;Nakamura T;Oshimura M
微细胞介导的染色体转移（MMCT）是一种通过微细胞融合将染色体从供体细胞转移到受体细胞的技术。聚乙二醇（PEG）通常用作融合剂，并且在各种遗传研究中非常成功。然而，PEG并不适用于所有类型的受体细胞，因为其细胞类型依赖性毒性。PEG的细胞毒性限制了微细胞杂交的产量到低水平（10-6至10-5/受体细胞）。为了充分利用MMCT的潜力，需要开发一种毒性更小、更有效、易于操作的融合方案。用编码减毒麻疹病毒（MV）Edmonston株的血凝素（H）和融合（F）蛋白的基因转染携带人人工染色体（HAC）的微细胞供体CHO细胞。CHO转染子和MV感染能力的人纤维肉瘤细胞（HT 1080）的混合培养物形成多核合胞体，表明MV-H/F在CHO细胞中的功能性表达。制备微细胞并将其应用于HT 1080细胞、人永生化间充质干细胞（hiMSC）和原代成纤维细胞。在用杀稻瘟菌素靶向HAC上标记的选择标记基因的培养物中选择后，出现了耐药细胞。通过计数每个实验中获得的稳定克隆的总数来确定融合效率。通过FISH分析证实了微细胞杂交体中HAC的保留。三种受体细胞系显示出不同的融合效率，其取决于作为MV受体的CD 46的细胞表面表达水平。在HT 1080和hiMSC中，观察到的最大效率分别是使用常规PEG融合的50倍和100倍。然而，PEG诱导的与HFL 1融合的低效率并没有被MV融合子改善。MV包膜蛋白的异位表达提供了一种有效的受体细胞导向的MMCT方案，促进了基因功能和遗传校正研究的广泛应用。
Microcell-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. Microcell 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. Ectopic expression of MV envelope proteins provides an efficient recipient cell-oriented MMCT protocol, facilitating extensive applications for studies of gene function and genetic corrections.
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CTCFL/BORIS是一种与甲基化无关的DNA结合蛋白，优先结合父亲H19差异甲基化区域。
影响因子：
11.2
作者：
Nguyen P;Cui H;Bisht KS;Sun L;Patel K;Lee RS;Kugoh H;Oshimura M;Feinberg AP;Gius D
通讯作者：
Gius D
影响因子：
12.4
作者：
Hoshiya, Hidetoshi;Kazuki, Yasuhiro;Oshimura, Mitsuo
通讯作者：
Oshimura, Mitsuo
影响因子：
2.2
作者：
GRAMMATIKOS, SI;SUBBAIAH, PV;MILLER, WM
通讯作者：
MILLER, WM
影响因子：
3.5
作者：
Quaye, Lydia;Dafou, Dimitra;Gayther, Simon A.
通讯作者：
Gayther, Simon A.
影响因子：
7.8
作者：
ROOS, DS;CHOPPIN, PW
通讯作者：
CHOPPIN, PW