Rhodes K, Hall K, Lee K E, Razzaghi H, Breindl M
Department of Biology and Molecular Biology Institute, San Diego University, CA 92182, USA.
Gene Expr. 1996;6(1):35-44.
An in vitro differentiation system utilizing retinoic acid (RA) treatment of pluripotent murine P19 embryonal carcinoma (EC) cells, which can be induced to differentiate into various cell types, was optimized for maximal induction of alpha 1 type I collagen (Col1a1) gene expression. Differentiation was associated with apoptotic death of the majority of cells, indicating that this in vitro system faithfully mimics the in vivo differentiation process. Col1a1 mRNA became detectable by RNase protection assay after 3 days of RA treatment and, after 6 days, reached a level comparable to that in NIH 3T3 fibroblasts. After induction of differentiation the Col1a1 gene remained transcriptionally active for extended periods of time even in the absence of RA. A minigene version of the murine Col1a1 gene was constructed that contains all of the so far known Col1a1 regulatory elements. This construct exhibited the correct expression pattern in stable transfection experiments: it was expressed in fibroblasts, but not in undifferentiated P19 EC cells, and it was transcriptionally activated after induction of differentiation. This experimental system should be a useful tool for dissecting the molecular mechanisms involved in the developmental activation and stage- and tissue-specific expression of the murine Col1a1 gene.
利用视黄酸（RA）处理多能性小鼠P19胚胎癌细胞（可诱导分化为多种细胞类型）建立了一种体外分化系统，该系统经优化可最大程度地诱导α1Ⅰ型胶原（Col1a1）基因表达。分化与大多数细胞的凋亡死亡相关，表明该体外系统忠实地模拟了体内分化过程。RA处理3天后，通过核糖核酸酶保护试验可检测到Col1a1 mRNA，6天后，其水平达到与NIH 3T3成纤维细胞相当的水平。诱导分化后，即使在没有RA的情况下，Col1a1基因仍能长时间保持转录活性。构建了包含所有目前已知的Col1a1调控元件的小鼠Col1a1基因的微型基因版本。该构建体在稳定转染实验中表现出正确的表达模式：在成纤维细胞中表达，但在未分化的P19 EC细胞中不表达，并且在诱导分化后被转录激活。该实验系统应是剖析参与小鼠Col1a1基因发育激活以及阶段和组织特异性表达的分子机制的有用工具。
