Department of Cell and Developmental Biology, University of Colorado Denver Aurora, CO, USA ; Charles C. Gates Regenerative Medicine and Stem Cell Biology Program, University of Colorado Denver Aurora, CO, USA.
Department of Cell and Developmental Biology, University of Colorado Denver Aurora, CO, USA.
Front Genet. 2014 Sep 8;5:310. doi: 10.3389/fgene.2014.00310. eCollection 2014.
Longevity is correlated with stress resistance in many animal models. However, previous efforts through the boosting of the antioxidant defense system did not extend life span, suggesting that longevity related stress resistance is mediated by other uncharacterized pathways. We have developed a high-throughput platform for screening and rapid identification of novel genetic mutants in the mouse that are stress resistant. Selection for resistance to stressors occurs in mutagenized mouse embryonic stem (ES) cells, which are carefully treated so as to maintain pluripotency for mouse production. Initial characterization of these mutant ES cells revealed mutations in Pigl, Tiam1, and Rffl, among others. These genes are implicated in glycosylphosphatidylinositol biosynthesis, NADPH oxidase function, and inflammation. These mutants: (1) are resistant to two different oxidative stressors, paraquat and the omission of 2-mercaptoethanol, (2) have reduced levels of endogenous reactive oxygen species (ROS), (3) are capable of generating live mice, and (4) transmit the stress resistance phenotype to the mice. This strategy offers an efficient way to select for new mutants expressing a stress resistance phenotype, to rapidly identify the causative genes, and to develop mice for in vivo studies.
在许多动物模型中，长寿与抗应激能力相关。然而，通过增强抗氧化防御系统来提高抗应激能力的先前努力并没有延长寿命，这表明与长寿相关的抗应激能力是由其他未被描述的途径介导的。我们开发了一种高通量筛选和快速鉴定对压力有抗性的新型遗传突变体的方法，该方法适用于小鼠。在经过诱变的小鼠胚胎干细胞（ES 细胞）中进行应激选择，这些细胞经过精心处理以保持其产生小鼠的多能性。对这些突变型 ES 细胞的初步特征分析显示，Pigl、Tiam1 和 Rffl 等基因发生了突变。这些基因涉及糖基磷脂酰肌醇生物合成、NADPH 氧化酶功能和炎症。这些突变体：（1）对两种不同的氧化应激物，百草枯和 2-巯基乙醇的缺失具有抗性；（2）具有较低水平的内源性活性氧（ROS）；（3）能够产生活的小鼠；（4）将抗应激表型传递给小鼠。这种策略为选择表达抗应激表型的新突变体提供了一种有效方法，能够快速鉴定致病基因，并开发用于体内研究的小鼠。
