Biolistic transfection and morphological analysis of cultured sympathetic neurons
Biolistic transfection and morphological analysis of cultured sympathetic neurons
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DOI：
10.1016/s0165-0270(01)00473-3
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发表时间：
2002-01-15
影响因子：
3
通讯作者：
Pfaller, K
中科院分区：
文献类型：
--
作者：
Klimaschewski, L;Nindl, W;Pfaller, K
我们已经开发了一种将外源基因引入有丝分裂后周围神经元的方案，并通过计算机辅助形态测定法对其进行后续分析。生物学方法的应用导致了转染。大约10%分离的交感神经细胞。包裹着质粒DNA的金颗粒通过手持“基因枪”进入交感神经细胞核。转染。选择每次实验的微载体上载量为0.7 mg金，DNA上载比为每mg金2杯DNA，以优化效率。基因枪经过改良，可以最大限度地减少压力引起的细胞损伤。用编码增强型绿色荧光蛋白（EGFP）或红色荧光蛋白（DsRed）的质粒转染神经元，作为细胞体、轴突突起和生长锥（包括板足和丝足）的合适标记。描述了使用METAMORPH（TM）软件进行定量形态测量图像分析的详细程序。通过比较神经生长因子（NGF）和二丁基环腺苷单磷酸（dbcAMP）对玻璃底皿上转染的交感神经元的神经突生长的影响，证明了该方案。(C) 2002 Elsevier Science B.V.版权所有
We have developed a protocol for introducing foreign genes into postmitotic peripheral neurons and their subsequent analysis by computer-assisted morphometry. The application of the biolistic approach results in transfection. of approximately 10% of dissociated sympathetic neurons. Gold particles coated with plasmid DNA are propelled into sympathetic neuronal nuclei using a hand-held 'gene gun'. Transfection. efficiency is optimized by choosing a microcarrier loading quantity of 0.7 mg of gold per experiment and a DNA loading ratio of 2 mug DNA per mg of gold. The gene gun is modified to minimize pressure-induced cell damage. Neurons are transfected with plasmids encoding enhanced green fluorescent protein (EGFP) or red fluorescent protein (DsRed), which serve as suitable markers for the cell body, axonal processes and growth cones including lamellipodia and filopodia. A detailed routine for quantitative morphometric image analysis using METAMORPH(TM) software is described. The protocol is demonstrated by comparing the effects of nerve growth factor (NGF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) on neurite outgrowth of transfected sympathetic neurons plated on glass floor dishes. (C) 2002 Elsevier Science B.V. All rights reserved.