Condreay J P, Witherspoon S M, Clay W C, Kost T A
Glaxo Wellcome Research Institute, P.O. Box 13398, Research Triangle Park, NC 27709, USA.
Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):127-32. doi: 10.1073/pnas.96.1.127.
Recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by inclusion of a dominant selectable marker in the viral vector, cell lines can be derived that stably express recombinant genes. A virus was constructed containing two expression cassettes controlled by constitutive mammalian promoters: the cytomegalovirus immediate early promoter/enhancer directing expression of green fluorescent protein and the simian virus 40 (SV40) early promoter controlling neomycin phosphotransferase II. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. In addition to commonly used transformed cell lines such as HeLa, CHO, Cos-7, and 293, this list includes primary human keratinocytes and bone marrow fibroblasts. In all cases, addition of butyrate or trichostatin A (a selective histone deacetylase inhibitor) to transduced cells markedly enhanced the levels of reporter protein expression observed. When transduced cells are put under selection with the antibiotic G418, cell lines can be obtained at high frequency that stably maintain the expression cassettes of the vector DNA and exhibit stable, high-level expression of the reporter gene. Stably transduced derivatives have been selected from a substantial number of different cell types, suggesting that stable lines can be derived from any cell type that exhibits transient expression.
重组杆状病毒可作为基因传递载体，用于在多种哺乳动物细胞类型中瞬时表达重组蛋白。此外，通过在病毒载体中加入显性选择标记，可以获得稳定表达重组基因的细胞系。构建了一种病毒，其包含两个由组成型哺乳动物启动子控制的表达盒：巨细胞病毒立即早期启动子/增强子指导绿色荧光蛋白的表达，猿猴病毒40（SV40）早期启动子控制新霉素磷酸转移酶II。使用这种病毒，在许多人类、灵长类和啮齿类来源的细胞类型中观察并测量到了有效的基因传递和表达。除了常用的转化细胞系如HeLa、CHO、Cos-7和293外，这个列表还包括原代人角质形成细胞和骨髓成纤维细胞。在所有情况下，向转导细胞中添加丁酸盐或曲古抑菌素A（一种选择性组蛋白脱乙酰酶抑制剂）可显著提高观察到的报告蛋白表达水平。当用抗生素G418对转导细胞进行筛选时，可以高频获得稳定维持载体DNA表达盒并表现出报告基因稳定、高水平表达的细胞系。已经从大量不同的细胞类型中筛选出了稳定转导的衍生物，这表明稳定细胞系可以从任何表现出瞬时表达的细胞类型中获得。
