Fluorescent methotrexate labeling and flow cytometric analysis of cells containing low levels of dihydrofolate reductase.
Fluorescent methotrexate labeling and flow cytometric analysis of cells containing low levels of dihydrofolate reductase.
复制标题
对含有低水平二氢叶酸还原酶的细胞进行荧光甲氨蝶呤标记和流式细胞术分析。
DOI：
--
复制
发表时间：
1986
影响因子：
4.8
通讯作者：
Geoffrey M. Wahlg
中科院分区：
文献类型：
--
作者：
Patrick Gaudray;Joseph Trotter;Geoffrey M. Wahlg
先前的研究已经证明了流式细胞术在分析二氢叶酸还原酶（EC 1.5.1.3）基因扩增中的有用性。然而，这种用于分析单个细胞中基因表达的强大且潜在敏感的方法尚未广泛使用。这部分是由于生产荧光甲氨蝶呤（Fluo-MTX）的困难，这是需要在体内标记二氢叶酸还原酶，产率高于1%，并有足够的纯度，以提供低非特异性背景的公布的程序。我们对Fluo-MTX的合成进行了显著改进，以20%的收率快速获得色谱纯的产品。此外，我们发现细胞体积是一个变量，这使得直接比较细胞系之间的荧光强度变得困难。为了解决这个问题，我们改进了流式细胞仪分析，以测量单个细胞的荧光比强度。对基因转移常用的各种细胞的调查显示，它们用Fluo-MTX标记的效率存在显著差异，这似乎是由于它们转运该试剂的能力存在差异。
Previous studies have demonstrated the usefulness of flow cytometry in the analysis of dihydrofolate reductase (EC 1.5.1.3) gene amplification. However, this powerful and potentially sensitive method for analyzing gene expression in individual cells has not seen widespread use. This is due in part to the difficulty of producing fluorescent methotrexate (Fluo-MTX), which is needed to label dihydrofolate reductase in vivo, in yields higher than 1% and of sufficient purity to give low nonspecific backgrounds by the published procedures. We have significantly improved the synthesis of Fluo-MTX to obtain rapidly a chromatographically pure product in 20% yields. In addition, we have found that cell volume is a variable which makes direct comparisons of fluorescence intensity between cell lines difficult. In order to circumvent this problem, we have improved flow cytometric analysis to measure the fluorescence specific intensity of individual cells. A survey of various cells commonly used for gene transfer shows a significant variability in the efficiency with which they are labeled with Fluo-MTX, which appears to be due to variations in their ability to transport this reagent.