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首页> 外文期刊>Molecular and Cellular Biology >Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension.
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Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension.

机译：克隆的DNA高效转移到人二倍体细胞中：原生质体在悬浮液中融合。

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摘要

A method for fusion of protoplasts bearing amplified plasmids and human diploid fibroblasts or other cell types in suspension is described. Transient expression of plasmid-encoded proteins occurs in up to 50% of the human cells, as demonstrated for simian virus 40 T antigen by immunofluorescence and the Escherichia coli xanthine-guanine phosphoribosyl transferase by autoradiography. In contrast, frequencies of stable transformants were similar to those obtained by the CaPO4 coprecipitation technique. However, experiments with both methods involving the recombinant pRSVneo (in which the Rous sarcoma virus long terminal repeat regulates expression of the antibiotic-inactivating aminoglycoside phosphotransferase) revealed a much higher frequency of colonies in G418 selective medium with constructions in which the early region of simian virus 40 DNA was present as well. We propose a role for the simian virus 40 T antigen in enhancing stable transformation in this system.
机译：描述了一种在悬浮液中融合带有扩增质粒和人二倍体成纤维细胞或其他细胞类型的原生质体的方法。质粒编码蛋白的瞬时表达在多达50％的人类细胞中发生，如通过免疫荧光检测的猿猴病毒40 T抗原和通过放射自显影检测的大肠杆菌黄嘌呤-鸟嘌呤磷酸核糖基转移酶。相反，稳定转化体的频率与通过CaPO4共沉淀技术获得的频率相似。然而，两种方法的实验均涉及重组pRSVneo（其中劳斯肉瘤病毒的长末端重复序列调节了灭活抗生素的氨基糖苷磷酸转移酶的表达），发现在G418选择性培养基中菌落的发生频率要高得多，其构型中猿猴的早期区域病毒40 DNA也存在。我们提出了猿猴病毒40 T抗原在增强该系统中稳定转化中的作用。

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