Transfection of a reporter plasmid into cultured cells by sonoporation in vitro
Transfection of a reporter plasmid into cultured cells by sonoporation in vitro
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DOI：
10.1016/s0301-5629(97)00025-2
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发表时间：
1997-01-01
影响因子：
2.9
通讯作者：
Miller, DL
中科院分区：
文献类型：
--
作者：
Bao, SP;Thrall, BD;Miller, DL
将培养的中国仓鼠卵巢细胞在无菌的4.5-mL聚乙烯室中暴露于2.25-MHz超声，并测试细胞裂解、声致穿孔和DNA转染。加入10%的Albunex（R），一种基于气体体的超声造影剂，以确保空化成核，并将室以60 rpm旋转以在1-min暴露期间促进空化活性，对于低至0.1MPa的空间峰值压力幅度，观察到一些细胞对大荧光葡聚糖分子的摄取，这表明瞬时透化和重新密封，即，在0.2MPa时细胞发生明显的溶解，在高于表观空化阈值时细胞溶解迅速增加（使用H2 O2产生试验）约0.4MPa空间峰值压力振幅。在DNA转染试验中，在暴露期间将20 μ g/mL荧光素酶报告质粒加入悬浮液中，暴露2天后测定细胞的增殖能力和荧光素酶基因表达。细胞增殖在空化阈值以上大大降低，荧光素酶产生对于0.20-MPa暴露是显著的，在0.8-MPa暴露下达到0.33 ng/10（6）个细胞。暴露于补充有血清的培养基中的细胞的荧光素酶产量大于暴露于无血清培养基中的细胞。在对数期或培养的稳定期收获暴露的细胞得到相似的增殖和转染结果，当从悬浮液中省略Albunex并且管不旋转时，效果基本上消失。因此，在旋转管暴露系统中通过超声空化的声致穿孔产生质粒转染，随后瞬时基因表达。(C)1997年世界医学与生物学超声联合会。
Cultured Chinese hamster ovary cells were exposed to 2.25-MHz ultrasound in sterile 4.5-mL polyethylene chambers and tested for cell lysis, sonoporation and DNA transfection, Ten percent of Albunex(R), a gas-body-based ultrasound contrast agent, was added to ensure cavitation nucleation, and the chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures, Uptake of large fluorescent dextran molecules by some cells was observed for spatial peak pressure amplitudes as low as 0.1 MPa, which indicates transient permeabilization and resealing, i.e., sonoporation, of these cells during exposure, Significant lysis occurred for 0.2 MPa, and increased rapidly for exposures above the apparent cavitation threshold (using the H2O2 production test) of about 0.4 MPa spatial peak pressure amplitude, In the DNA transfection tests, 20 mu g/mL luciferase reporter plasmid was added to the suspension during exposure, and cells were assayed for proliferation ability and luciferase gene expression 2 days after exposure, Cell proliferation was greatly reduced above the cavitation threshold, Luciferase production was significant for 0.20-MPa exposure, and reached 0.33 ng per 10(6) cells at 0.8-MPa exposure, The luciferase production was greater for cells exposed in medium supplemented with serum than for cells exposed in serum-free medium, Cells harvested for exposure either in the log phase or in the stationary phase of culture gave similar proliferation and transfection results, The effects essentially disappeared when the Albunex was omitted from the suspension and the tube was not rotated, Thus, sonoporation by ultrasonic cavitation in the rotating tube exposure system yields plasmid transfection with subsequent transient gene expression. (C) 1997 World Federation for Ultrasound in Medicine & Biology.