Nucleofection of muscle-derived stem cells and myoblasts with φC31 integrase:: Stable expression of a full-length-dystrophin fusion gene by human myoblasts
Nucleofection of muscle-derived stem cells and myoblasts with φC31 integrase:: Stable expression of a full-length-dystrophin fusion gene by human myoblasts
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DOI：
10.1016/j.ymthe.2004.05.034
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发表时间：
2004-10-01
影响因子：
12.4
通讯作者：
Tremblay, JP
中科院分区：
文献类型：
--
作者：
Quenneville, SP;Chapdelaine, P;Tremblay, JP
离体基因疗法通过将肌营养不良蛋白基因转染到患者自身的肌源性前体细胞中，然后进行移植，为杜氏肌营养不良症提供了一种潜在的治疗方法。我们使用核转染将编码增强型绿色荧光蛋白 (eGFP) 或 eGFP-肌营养不良蛋白融合蛋白的 DNA 质粒和噬菌体 phiC31 整合酶引入生肌细胞中，并将这些基因整合到基因组中有限数量的位点中。使用表达 eGFP 的质粒，我们转染了 50% 的小鼠肌肉干细胞系和 60% 的正常人成肌细胞。由于转基因的位点特异性整合，表达 phiC31 整合酶的质粒和含有 attB 序列的 eGFP 表达质粒的共核转染产生了 15 倍频率的稳定表达。 phiC31整合酶质粒和含有attB序列和eGFP-全长肌营养不良蛋白融合蛋白基因的大质粒的共核转染产生了荧光人类成肌细胞，培养1个月后，这些成肌细胞能够形成更强烈的荧光肌管。结合核转染和 phiC31 整合酶的非病毒方法最终可能允许将转基因细胞安全地自体移植给患者。
Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient's own myogenic precursor cells, followed by transplantation. We used nucleofection to introduce DNA plasmids coding for enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. Using a plasmid expressing eGFP, we transfected 50% of a mouse muscle-derived stem cell line and 60% of normal human myoblasts. Co-nucleofection of a plasmid expressing the phiC31 integrase and an eGFP expression plasmid containing an attB sequence produced 15 times more frequent stable expression, because of site-specific integration of the transgene. Co-nucleofection of the phiC31 integrase plasmid and a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after 1 month of culture. A nonviral approach combining nucleofection and the phiC31 integrase may eventually permit safe autotransplantation of genetically modified cells to patients.