Selection of glucocorticoid-resistant mutations from an AtT-20 cell line containing a glucocorticoid-regulated selectable transgene.
Selection of glucocorticoid-resistant mutations from an AtT-20 cell line containing a glucocorticoid-regulated selectable transgene.
复制标题
从含有糖皮质激素调节的选择性转基因的 AtT-20 细胞系中选择糖皮质激素抗性突变。
DOI：
10.1006/bbrc.1995.1464
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发表时间：
1995
影响因子：
3.1
通讯作者：
VerHoeven,R
中科院分区：
文献类型：
--
作者：
Harrison3rd,RW;Lippman,SS;VerHoeven,R
AtT-20/IDG8 细胞含有稳定转染的选择性基因新霉素磷酸转移酶，处于负糖皮质激素调节下。因此，当在新霉素类似物 G418 和地塞米松同时存在的情况下培养时，AtT-20/IDG8 细胞无法生长。我们的假设是，能够在此类培养基中生长的突变 AtT-20/IDG8 细胞在糖皮质激素介导的 neorgene 调节中存在缺陷。使用甲磺酸乙酯对 AtT-20/IDG8 细胞进行化学诱变，并在 G418 和地塞米松存在下进行克隆。获得了14个克隆，并证实它们都失去了糖皮质激素对新表达的控制。天然存在的基因阿片黑皮素原在亲本 AtT-20/1DG8 细胞中被糖皮质激素下调，而在 10 个突变系中，地塞米松也下调了该基因，这表明在这些细胞中，尽管 neor 的调节异常，该受体仍具有功能。在其他四个品系中，阿片黑皮质素原调节丢失，这也表明一般转录因子（例如受体）已被改变。这些结果表明，糖皮质激素介导的基因调控涉及多种因素，并且在将受调控的选择性基因插入先前不可选择性的细胞系中后，可以产生新的、信息丰富的突变。
AtT-20/IDG8 cells contain the stably transfected, selectable gene, neomycin phosphotransferase, under negative glucocorticoid regulation. Thus, when cultured in the simultaneous presence of the neomycin analogue, G418, and dexamethasone, AtT-20/IDG8 cells fail to grow. Our hypothesis was that mutated AtT-20/IDG8 cells capable of growth in such medium would have a defect in the glucocorticoid-mediated regulation of the neorgene. AtT-20/IDG8 cells were chemically mutagenized using ethyl-methane sulfonate and cloned in the presence of G418 and dexamethasone. Fourteen clones were obtained and loss of glucocorticoid control of neorexpression was confirmed in them all. The naturally occurring gene, pro-opiomelanocortin, which is down-regulated by glucocorticoids in parent AtT-20/1DG8 cells, was down-regulated by dexamethasone in ten of the mutant lines, indicating that in those cells the receptor was functional in spite of aberrant regulation of neor. In the other four lines, pro-opiomelanocortin regulation was lost,also suggesting that a general transcription factor, such as the receptor, had been altered. These results indicate that multiple factors are involved in glucocorticoid-mediated gene regulation and that new, informative mutations can be produced after insertion of a regulated, selectable gene into a previously non-selectable cell line.