Pieper F R, Schaart G, Krimpenfort P J, Henderik J B, Moshage H J, van de Kemp A, Ramaekers F C, Berns A, Bloemendal H
Department of Biochemistry, University of Nijmegen, The Netherlands.
J Cell Biol. 1989 Mar;108(3):1009-24. doi: 10.1083/jcb.108.3.1009.
The coding region of the hamster desmin gene was fused to the 5' flanking sequences of the hamster vimentin gene and introduced into the germ line of mice. The expression of this intermediate filament gene construct (pVDes) was analyzed at the RNA and protein level in transgenic mice as well as in fibroblast cell lines and primary hepatocyte cultures derived from these mice. In all transgenic mice, the pVDes-encoded protein was coexpressed with mouse vimentin in a tissue-specific fashion and was indistinguishable from normal hamster desmin. Culturing of transgenic hepatocytes induced desmin expression indicating that 3.2 kbp of the vimentin gene 5' region regulates both tissue-specific and tissue culture-induced intermediate filament protein expression. Immunohistochemical staining and double-label immunoelectron microscopy of cultured transgenic fibroblasts showed that the pVDes protein assembled into intermediate filaments which colocalized with the mouse vimentin filaments. Endogenous vimentin RNA levels were not influenced by high-level pVDes expression. The coexpression of desmin and vimentin in nonmuscle cells did not result in detectable developmental, morphological, or physiological abnormalities.
将仓鼠结蛋白基因的编码区与仓鼠波形蛋白基因的5'侧翼序列融合,并导入小鼠生殖系。在转基因小鼠以及源自这些小鼠的成纤维细胞系和原代肝细胞培养物中,从RNA和蛋白质水平分析了这种中间丝基因构建体(pVDes)的表达。在所有转基因小鼠中,pVDes编码的蛋白质与小鼠波形蛋白以组织特异性方式共表达,并且与正常仓鼠结蛋白没有区别。转基因肝细胞的培养诱导了结蛋白表达,表明波形蛋白基因5'区域的3.2kbp调节组织特异性和组织培养诱导的中间丝蛋白表达。对培养的转基因成纤维细胞进行免疫组织化学染色和双标记免疫电子显微镜检查表明,pVDes蛋白组装成与小鼠波形蛋白丝共定位的中间丝。内源性波形蛋白RNA水平不受高水平pVDes表达的影响。结蛋白和波形蛋白在非肌肉细胞中的共表达未导致可检测到的发育、形态或生理异常。