Litzkas P, Jha K K, Ozer H L
Mol Cell Biol. 1984 Nov;4(11):2549-52. doi: 10.1128/mcb.4.11.2549-2552.1984.
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.
本文描述了一种用于将携带扩增质粒的原生质体与悬浮培养的人二倍体成纤维细胞或其他细胞类型进行融合的方法。通过免疫荧光检测猿猴病毒40 T抗原以及通过放射自显影检测大肠杆菌黄嘌呤 - 鸟嘌呤磷酸核糖转移酶表明，高达50%的人细胞中会出现质粒编码蛋白的瞬时表达。相比之下，稳定转化体的频率与通过磷酸钙共沉淀技术获得的频率相似。然而，使用涉及重组pRSVneo（其中劳氏肉瘤病毒长末端重复序列调节抗生素失活氨基糖苷磷酸转移酶的表达）的两种方法进行的实验表明，在含有猿猴病毒40 DNA早期区域的构建体的G418选择培养基中，菌落频率要高得多。我们提出猿猴病毒40 T抗原在该系统中增强稳定转化方面发挥作用。
