Wellmann H, Kaltschmidt B, Kaltschmidt C
Molecular Neurobiology Laboratory, Institute of Anatomy, Albert-Ludwigs-University, Freiburg, Germany.
J Neurosci Methods. 1999 Oct 15;92(1-2):55-64. doi: 10.1016/s0165-0270(99)00094-1.
DNA-transfer into postmitotic neurons or neuronal tissues has been a major problem in neurobiology. For this aim different methods have been used such as viral infection, microinjection, lipofection or calcium phosphate precipitation. However, using these techniques, very poor transfection efficiency was achieved except for virus-mediated gene transfer. Though viral infections are very efficient, this method is expensive and labor-intensive, especially when recombination is used to prepare viral vectors. Biolistic gene transfer of neurons represents another promising transfection technique. This technique was originally used to transfect plant cells and has been further developed for gene transfer into neurons or neuronal tissues. Up to now, only a few reports are available where successful biolistic gene transfer into neurons or neuronal tissues could be shown. Transfection efficiencies were only about 2%. Most of the previously published experiments were carried out under vacuum conditions using in-chamber gene gun types. Here we describe an improved method for efficient neuronal cell transfection using a hand-held gene gun. Expression vectors could be successfully transferred into dissociated cultured hippocampal neurons, PC12 cells, cultured cerebellar granule cells and cerebellar brain slices. In cerebellar granule cells and hippocampal neurons, transfection efficiencies of about 10% were reached.
将DNA导入有丝分裂后的神经元或神经组织一直是神经生物学中的一个主要问题。为此，人们使用了不同的方法，如病毒感染、显微注射、脂质体转染或磷酸钙沉淀法。然而，使用这些技术，除了病毒介导的基因转移外，转染效率非常低。虽然病毒感染效率很高，但这种方法昂贵且 labor-intensive，特别是在使用重组技术制备病毒载体时。神经元的生物弹道基因转移是另一种有前景的转染技术。该技术最初用于转染植物细胞，并已进一步发展用于将基因导入神经元或神经组织。到目前为止，只有少数报告表明成功地将生物弹道基因转移到神经元或神经组织中。转染效率仅约为2%。以前发表的大多数实验是在真空条件下使用腔内基因枪类型进行的。在这里，我们描述了一种使用手持式基因枪进行高效神经元细胞转染的改进方法。表达载体可以成功地转移到解离培养的海马神经元PC12细胞、培养的小脑颗粒细胞和小脑脑片中。在小脑颗粒细胞和海马神经元中，转染效率达到了约10%。
