Dierich A, Gaub M P, LePennec J P, Astinotti D, Chambon P
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Institut de Chimie Biologique, Faculté de Médecine, Strasbourg, France.
EMBO J. 1987 Aug;6(8):2305-12. doi: 10.1002/j.1460-2075.1987.tb02505.x.
A series of recombinant plasmids containing increasing lengths of the 5'-flanking promoter sequences of the chicken conalbumin and ovalbumin genes fused to the sequences coding for the SV40 T-antigen have been constructed. These recombinants were introduced into a variety of established cell lines and primary cultured cells by nuclear microinjection. Promoter activity was estimated by monitoring T-antigen synthesis by indirect immunofluorescence. We show that the microinjected ovalbumin and conalbumin promoter regions do not function in chicken fibroblasts, kidney cells and in a variety of non-chicken cells, irrespective of the presence of steroid hormone receptors. In contrast, these promoter regions are active in primary cultured chicken embryonic hepatocytes and oviduct tubular gland cells, suggesting the presence of cell-specific transcription factors in these cells. Unexpectedly, promoter sequences close to the TATA boxes of both the ovalbumin and conalbumin genes are sufficient to confer cell-specific expression. Most of the controls exerted on the ovalbumin and conalbumin promoters in the whole animal appear to be reproduced in vitro by nuclear microinjection of the chimeric genes into the primary cultured cells. However, the microinjected ovalbumin promoter is active in embryonic hepatocytes and thus escapes the regulation imposed on the corresponding inactive endogenous gene.
已构建了一系列重组质粒,这些质粒包含鸡伴清蛋白和卵清蛋白基因5'-侧翼启动子序列的长度不断增加的片段,并与编码SV40 T抗原的序列融合。通过核显微注射将这些重组体导入多种已建立的细胞系和原代培养细胞中。通过间接免疫荧光监测T抗原的合成来评估启动子活性。我们发现,无论是否存在类固醇激素受体,显微注射的卵清蛋白和伴清蛋白启动子区域在鸡成纤维细胞、肾细胞以及多种非鸡细胞中均无功能。相反,这些启动子区域在原代培养的鸡胚肝细胞和输卵管管状腺细胞中具有活性,这表明这些细胞中存在细胞特异性转录因子。出乎意料的是,靠近卵清蛋白和伴清蛋白基因TATA框的启动子序列足以赋予细胞特异性表达。通过将嵌合基因核显微注射到原代培养细胞中,在体外似乎重现了在整个动物中对卵清蛋白和伴清蛋白启动子施加的大多数调控。然而,显微注射的卵清蛋白启动子在胚胎肝细胞中具有活性,因此逃脱了对相应无活性内源性基因施加的调控。