Nanoparticles of compacted DNA transfect postmitotic cells
Nanoparticles of compacted DNA transfect postmitotic cells
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DOI：
10.1074/jbc.m305776200
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发表时间：
2003-08-29
影响因子：
4.8
通讯作者：
Cooper, MJ
中科院分区：
文献类型：
--
作者：
Liu, G;Li, DS;Cooper, MJ
电荷中性的DNA纳米颗粒已经被开发出来，其中DNA的单个分子被压缩到其最小可能的尺寸。我们推测，这些DNA纳米颗粒的小尺寸可能有助于有丝分裂后细胞中的基因转移，允许核摄取穿过25 nm的核膜孔。为了确定DNA纳米颗粒是否可以抑制非分裂细胞，用编码荧光素酶的DNA/脂质体混合物转染生长停滞的神经母细胞瘤和肝癌细胞。在这两种模型中，生长停滞的细胞被压实的DNA稳健地转染（比裸DNA多6，900 -360倍）。为了评估负责增强转染的机制，HuH-7细胞用编码增强的绿色荧光蛋白的裸质粒或压缩质粒显微注射。与裸DNA相比，细胞质显微注射DNA纳米颗粒产生了类似于10倍的转基因表达的改善;这种增强被核孔抑制剂小麦胚芽凝集素逆转。为了确定基因转移的大小上限，将各种大小的DNA纳米颗粒显微注射到细胞质中。当小椭圆体直径接近25 nm时，观察到转基因表达显著降低。总之，适当大小的DNA纳米颗粒通过穿过核膜孔有效地抑制生长停滞的细胞。
Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a similar to10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.