Effective Plasmid DNA and Small Interfering RNA Delivery to Diseased Human Brain Microvascular Endothelial Cells
Effective Plasmid DNA and Small Interfering RNA Delivery to Diseased Human Brain Microvascular Endothelial Cells
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有效的质粒 DNA 和小干扰 RNA 递送至患病人脑微血管内皮细胞
DOI：
10.1159/000342909
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发表时间：
2012
影响因子：
1.2
通讯作者：
Schubert-Unkmeir A
中科院分区：
文献类型：
--
作者：
Slanina H;Schmutzler M;Christodoulides M;Kim KS;Schubert-Unkmeir A
外源DNA或小干扰RNA（siRNA）的体外表达受到所用特定递送系统的显著影响。在这项研究中，我们评估了质粒DNA和siRNA转染到人脑微血管内皮细胞（HBMEC）和脑膜瘤细胞，构成血-脑脊液屏障，脑膜炎病原体的目标的效率。应用化学转染方法和各种脂质转染试剂，包括Lipofectamin™、FuGene™或jetPRIME®，以及物理转染方法和电穿孔技术。为了监测转染效率，用报告质粒pTagGFP 2-actin载体转染HBMEC和脑膜瘤细胞，并通过荧光显微镜和流式细胞术评估转染效率。我们建立了基于电穿孔的方案，使用Cell Line Nucleofector® Kit V，使用来自Lonza的Amaxa® Nucleofector® II系统和来自Invitrogen的氖®转染系统，分别产生高达41%和82%的绿色荧光蛋白阳性HBMEC。评价最佳转染溶液、脉冲程序和长度。我们进一步证明了脂质体转染是一种有效的方法来转染脑膜瘤细胞，转染效率约为81%。最后，我们应用成功的电穿孔方案将合成的SiRNA递送到HBMEC，并分析了肌动蛋白结合蛋白皮质粘附素在脑膜炎奈瑟菌疾病发生中的作用。
Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin inNeisseria meningitidispathogenesis.
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影响因子：
3.7
作者：
Slanina H;Hebling S;Hauck CR;Schubert-Unkmeir A
通讯作者：
Schubert-Unkmeir A
影响因子：
15.9
作者：
Doran, KS;Engelson, EJ;Nizet, V
通讯作者：
Nizet, V
DOI：
10.1385/0-89603-310-4:1
发表时间：
1995
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Weaver,JC
通讯作者：
Weaver,JC
影响因子：
15.9
作者：
Ring, A;Weiser, JN;Tuomanen, EI
通讯作者：
Tuomanen, EI
DOI：
10.1073/pnas.79.2.422
发表时间：
1982-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
LOYTER, A;SCANGOS, GA;RUDDLE, FH
通讯作者：
RUDDLE, FH