Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells
Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells
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用于哺乳动物细胞中高频整合、表达和拯救基因的腺相关病毒载体
DOI：
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发表时间：
1985
影响因子：
5.3
通讯作者：
B. Carter
中科院分区：
文献类型：
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作者：
J. Tratschin;I. L. Miller;M. G. Smith;B. Carter
我们描述了腺相关病毒（AAV）载体的构建，其中原核基因neo的编码序列在主要AAV启动子p40的控制下表达。该AAV-neo载体通过选择对抗生素遗传霉素（G418）具有抗性的细胞，允许neo作为哺乳动物细胞中的显性选择标记物稳定表达。当通过DNA转染程序将载体引入人（293或HeLa）细胞系时，获得稳定的遗传霉素抗性集落。当载体首先被包装到AAV颗粒中，然后通过颗粒感染引入细胞中时，以比通过DNA转染获得的那些更高的频率获得遗传霉素抗性细胞。在遗传霉素抗性细胞中，AAV-neo载体以低拷贝数整合，并且可以通过随后用野生型AAV和辅助腺病毒感染或在某些情况下通过单独用腺病毒感染来拯救。然后可以从Hirt裂解物中回收拯救的AAV-neo载体作为扩增的未整合DNA。这些结果表明，AAV可用作转导病毒载体，用于外源基因在哺乳动物细胞中的稳定整合和表达。高频率的整合和拯救整合载体的能力表明，该载体系统可能是有用的从cDNA文库中选择基因。该载体也可用于将基因导入细胞中，所述细胞在诸如涉及使用CaPO 4或DEAE-葡聚糖的程序中难以转染。
We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.