A novel human artificial chromosome vector provides effective cell lineage-specific transgene expression in human mesenchymal stem cells
A novel human artificial chromosome vector provides effective cell lineage-specific transgene expression in human mesenchymal stem cells
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DOI：
10.1634/stemcells.2005-0021
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发表时间：
2005-11-01
期刊：
影响因子：
5.2
通讯作者：
Oshimura, M
中科院分区：
文献类型：
--
作者：
Ren, XY;Katoh, M;Oshimura, M
间充质干细胞（MSC）有望用于成人干细胞介导的基因治疗。干细胞介导的基因治疗的主要目的之一是开发载体，其将允许治疗基因在特化细胞的生理调节下沿着分化的适当水平的表达。人类人工染色体（HACs）作为独立的染色体稳定地存在于宿主细胞中，不存在常规转基因的潜在插入突变问题。因此，HAC已被提议作为细胞介导的基因治疗的替代工具。在此之前，我们构建了一种新的HAC，称为21 Δ pq HAC，具有loxP位点，其中环状DNA可以通过Cre/loxP系统可重复地插入。我们在此评估了通过21 Δ pq HAC载体使用具有成骨、成软骨和成脂分化潜力的MSC细胞系hiMSC的体外分化系统进行谱系特异性转基因表达的可行性。将由成骨谱系特异性骨桥蛋白（OPN）基因的启动子驱动的增强型绿色荧光蛋白（EGFP）基因插入到21 Delta pq HAC上，然后转移到hiMSC中。表达盒两侧是鸡HS 4绝缘子，以阻断相邻耐药基因的启动子干扰。EGFP基因在hiMSC中特异性表达，hiMSC与内源性OPN基因的转录协同分化为骨细胞，但在成脂分化诱导后或在非诱导培养中不表达。这些结果表明，HAC载体的使用适用于干细胞介导的基因治疗中转基因的调节表达。STEM C ELLS 2005;23：1608-1616.
Mesenchymal stem cells (MSCs) hold promise for use in adult stem cell-mediated gene therapy. One of the major aims of stem cell-mediated gene therapy is to develop vectors that will allow appropriate levels of expression of therapeutic genes along differentiation under physiological regulation of the specialized cells. Human artificial chromosomes (HACs) are stably maintained as independent chromosomes in host cells and should be free from potential insertional mutagenesis problems of conventional transgenes. Therefore, HACs have been proposed as alternative implements to cell-mediated gene therapy. Previously, we constructed a novel HAC, termed 21 Delta pq HAC, with a loxP site in which circular DNA can be reproducibly inserted by the Cre/loxP system. We here assessed the feasibility of lineage-specific transgene expression by the 21 Delta pq HAC vector using an in vitro differentiation system with an MSC cell line, hiMSCs, which has potential for osteogenic, chondrogenic, and adipogenic differentiation. An enhanced green fluorescent protein (EGFP) gene driven by a promoter for osteogenic lineage-specific osteopontin (OPN) gene was inserted onto the 21 Delta pq HAC and then transferred into hiMSC. The expression cassette was flanked by the chicken HS4 insulators to block promoter interference from adjacent drug-resistant genes. The EGFP gene was specifically expressed in the hiMSC that differentiated into osteocytes in coordination with the transcription of endogenous OPN gene but was not expressed after adipogenic differentiation induction or in noninduction culture. These results suggest that use of the HAC vector is suitable for regulated expression of transgenes in stem cell-mediated gene therapy. STEM C ELLS 2005;23:1608-1616.