HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes.
HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes.
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HSV-1 扩增子载体是骨骼肌成肌细胞和肌管的高效基因递送系统。
DOI：
10.1152/ajpcell.2000.278.3.c619
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Allen,PD
中科院分区：
文献类型：
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作者：
Wang,Y;Fraefel,C;Protasi,F;Moore,RA;Fessenden,JD;Pessah,IN;DiFrancesco,A;Breakefield,X;Allen,PD
由于使用磷酸钙或阳离子脂类技术对成肌细胞或肌管的低(5%)的转染率，使肌肉细胞中RyR1结构功能的分析变得困难。我们将15.3kb的RyR1全长基因插入到单纯疱疹病毒1型(HSV-1)扩增载体pHSVPrPUC中，插入启动子序列与HSV-1 DNA切割/包装信号(PAC)之间。用表达绿色荧光蛋白的两个扩增片段pHSVGN和PHSVGRyR1进行荧光激活细胞分选分析。用辅助病毒包装系统将所有扩增片段包装成HSV-1病毒颗粒，获得106个转导载体单位/毫升，HSVRyR1、HSVGRyR1和HSVGN病毒粒子有效地转导小鼠成肌细胞和肌管，在多次感染下70-90%的细胞表达所需产物。肌管产生了足够的蛋白质，可以从10个100毫米的培养皿中进行单通道分析。在大多数情况下，这种方法可以排除研究RyR1结构功能的永久转染体的需要。
Analysis of RyR1 structure function in muscle cells is made difficult by the low (<5%) transfection efficiencies of myoblasts or myotubes using calcium phosphate or cationic lipid techniques. We inserted the full-length 15.3-kb RyR1 cDNA into a herpes simplex virus type 1 (HSV-1) amplicon vector, pHSVPrPUC between theori/IE 4/5 promoter sequence and the HSV-1 DNA cleavage/packaging signal (pac). pHSVGN and pHSVGRyR1, two amplicons that expressed green fluorescent protein, were used for fluorescence-activated cell sorter analysis of transduction efficiency. All amplicons were packaged into HSV-1 virus particles using a helper virus-free packaging system and yielded 106transducing vector units/ml. HSVRyR1, HSVGRyR1, and HSVGN virions efficiently transduced mouse myoblasts and myotubes, expressing the desired product in 70–90% of the cells at multiplicity of infection 5. The transduced cells appeared healthy and RyR1 produced by this method was targeted properly and restored skeletal excitation-contraction coupling in dyspedic myotubes. The myotubes produced sufficient protein to allow single-channel analyses from as few as 10 100-mm dishes. In most cases this method could preclude the need for permanent transfectants for the study of RyR1 structure function.