Southern P J, Blount P, Oldstone M B
Nature. 1984;312(5994):555-8. doi: 10.1038/312555a0.
Nucleic acid hybridization techniques have contributed significantly to the understanding of gene organization, regulation and expression. In the context of persistent or latent viral infections, hybridization with specific labelled probes represents the most sensitive assay presently available for detection of viral genomes. During the course of persistence, viral genomes may exist in multiple and yet quite segregated areas in an infected host, but examination of all tissues remains difficult and time-consuming. At present, the application of hybridization assays to in vivo infections requires either chemical extraction of nucleic acid coupled with dot-blot and gel transfer techniques or in situ hybridization to cryostat tissue sections. In both cases, selected tissues must be removed by dissection before analysis. We describe here a procedure which allows efficient and reproducible screening of all tissue in an infected host. Our technique allows detection of viral genetic material in whole-body sections of infected mice, and provides the first evidence in vivo for accumulation of viral genetic material with a parallel decrease in infectious virus during persistent virus infection. This technique should be widely applicable to studies of developmental regulation of gene expression, for monitoring locations of gene expression in transgenic mice and for analysis of molecular mechanisms in pathogenesis.
核酸杂交技术对理解基因的组织、调控和表达有重大贡献。在持续性或潜伏性病毒感染的情况下，用特定的标记探针进行杂交是目前检测病毒基因组最灵敏的方法。在持续性感染过程中，病毒基因组可能存在于受感染宿主的多个且相当分散的区域，但检查所有组织仍然困难且耗时。目前，将杂交检测应用于体内感染，要么需要对核酸进行化学提取并结合斑点印迹和凝胶转移技术，要么需要对冷冻切片组织进行原位杂交。在这两种情况下，分析之前都必须通过解剖取出选定的组织。我们在此描述一种方法，该方法能对受感染宿主的所有组织进行高效且可重复的筛查。我们的技术能检测感染小鼠全身切片中的病毒遗传物质，并首次在体内证明在持续性病毒感染期间病毒遗传物质的积累以及感染性病毒的平行减少。该技术应广泛应用于基因表达的发育调控研究、监测转基因小鼠中基因表达的位置以及分析发病机制中的分子机制。
