Bhat K, McBurney M W, Hamada H
Faculty of Medicine, Memorial University of Newfoundland, St. John's, Canada.
Mol Cell Biol. 1988 Aug;8(8):3251-9. doi: 10.1128/mcb.8.8.3251-3259.1988.
Chromosomal loci that are specifically active in embryonal carcinoma stem cells were cloned from the mouse genome by functional selection. P19 cells, a pluripotent embryonal carcinoma cell line, were transfected with an enhancer trap (a plasmid containing an enhancerless inactive neo gene), and NEO+ transformants were isolated. All of the NEO+ cell lines retained pluripotency and expressed the neo gene. When the cells were induced to differentiate, most of the cell lines continued to express the neo gene, while the neo gene in some cell lines became repressed. From the latter group of cell lines, we have cloned the integrated neo gene plus the flanking cellular DNA sequences. Three of the six cloned DNAs possessed a high NEO+-transforming activity in undifferentiated P19 cells. Among these three, two (015 and 052) were inactive in differentiated P19 cells and NIH 3T3 cells, while the remaining one was active in these differentiated cells. Deletion analysis suggested that both 015 and 052 contain two regulatory elements (promoter and enhancer) of cellular DNA origin. The putative enhancer and promoter are separated by at least 6 kilobases in 015 and 1 kilobase in 052. Therefore, 015 and 052 cloned fragments contain regulatory DNA elements that are specifically active in the embryonal carcinoma stem cells.
通过功能筛选从小鼠基因组中克隆出在胚胎癌细胞干细胞中特异性活跃的染色体位点。将多能胚胎癌细胞系P19细胞用增强子陷阱（一种含有无增强子的无活性新霉素基因的质粒）转染，分离出NEO+转化体。所有NEO+细胞系都保持多能性并表达新霉素基因。当细胞被诱导分化时，大多数细胞系继续表达新霉素基因，而一些细胞系中的新霉素基因被抑制。从后一组细胞系中，我们克隆了整合的新霉素基因及其侧翼的细胞DNA序列。六个克隆的DNA中有三个在未分化的P19细胞中具有高NEO+转化活性。在这三个中，两个（015和052）在分化的P19细胞和NIH 3T3细胞中无活性，而另一个在这些分化细胞中有活性。缺失分析表明，015和052都含有细胞DNA来源的两个调控元件（启动子和增强子）。在015中，推定的增强子和启动子至少相隔6千碱基，在052中相隔1千碱基。因此，015和052克隆片段包含在胚胎癌细胞干细胞中特异性活跃的调控DNA元件。
