A new technique with calcium phosphate precipitate enhances efficiency of in vivo plasmid DNA gene transfer
A new technique with calcium phosphate precipitate enhances efficiency of in vivo plasmid DNA gene transfer
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DOI：
10.1254/jphs.fp0040504
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发表时间：
2005-02-01
影响因子：
3.5
通讯作者：
Kasugai, S
中科院分区：
文献类型：
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作者：
Kuroda, S;Kondo, H;Kasugai, S
质粒载体体内基因转移已在实验和临床上得到应用；然而，质粒载体的基因转移效率低是一个问题。我们推测磷酸钙沉淀（CaP）和质粒载体的结合可以解决这个问题，因为CaP可以稳定质粒DNA。在本研究中，我们使用编码增强型绿色荧光蛋白的质粒表达载体，并将该载体与CaP结合。然后，将该组合与牛 I 型去端胶原混合。将此混合物在磷酸盐缓冲盐水中温育后，当质粒DNA与CaP结合时，上清液中质粒DNA的量较低。此外，与CaP结合的质粒DNA在体外DNA酶消化中是稳定的。将带有或不带有CaP的质粒载体与去端肽胶原一起皮下移植或注射到大鼠股骨的骨髓中。然后，在共焦激光扫描显微镜下观察荧光并测量组织匀浆中的荧光强度。在这些动物实验中，当质粒 DNA 与 CaP 结合时，荧光很丰富。这些结果表明我们的配方，胶原蛋白/CaP/DNA，对于体内基因转移似乎是有效的。
In vivo gene transfer with plasmid vector has been applied experimentally and clinically; however, the low level of gene transfer efficiency with plasmid vector is a problem. We speculated that the combination of calcium phosphate precipitate (CaP) and plasmid vector could solve this problem because CaP stabilizes plasmid DNA. In the present study, we used a plasmid exression vector encoding enhanced green fluorescent protein and combined the vector with CaP. Then, this combination was mixed with bovine type I atelocollagen. After incubating this mixture in phosphate-buffered saline, the amount of the plasmid DNA in the supernatant was low when the plasmid DNA was combined with CaP. Furthermore, the plasmid DNA, which was combined with CaP, was stable in DNase digestion in vitro. The plasmid vector with or without CaP, together with the atelocollagen, was transplanted subcutaneously or injected in the bone marrow of the femurs of rats. Then, the fluorescence was observed under a confocal laser scanning microscope and the fluorescence intensity in the tissue homogenates was measured. In these animal experiments, the fluorescence was extensive when the plasmid DNA was combined with CaP. These results indicate that our formula, collagen/CaP/DNA, appeared efficient for in vivo gene transfer.