Screening for stress-resistance mutations in the mouse.

David Kitzenberg,Wallace S Chick,Kristina Williams,Thomas E Johnson,Xiaoyun Zhao,Michael Ludwig

doi:10.3389/fgene.2014.00310

Abstract

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.

Highlights

Stress resistance is a trait intimately linked to increased life span and health span in multiple species, including yeast, fruit flies, worms, and mice (Lithgow et al, 1995; Harshman and Haberer, 2000; Fabrizio et al, 2001; Johnson et al, 2002; Murakami et al, 2003)
CLONING OF piggyBac polyA TRAP VECTOR (PB-UPA) The unbiased polyA (UPA) trap vector was cloned by routine restriction digestion, or PCR, and ligation so that genetic elements were put in the order of splice-acceptor (SA) from the Bcl2 gene, bovine growth hormone polyA signal, phosphoglycerate kinase (PGK) promoter, neomycin resistant cassette, internal ribosomal entry site (IRES), and splice-donor (SD) from the Hprt gene
We are interested in identifying novel mouse genes that lead to resistance to multiple stressors

Summary

Introduction

Stress resistance is a trait intimately linked to increased life span and health span in multiple species, including yeast, fruit flies, worms, and mice (Lithgow et al, 1995; Harshman and Haberer, 2000; Fabrizio et al, 2001; Johnson et al, 2002; Murakami et al, 2003). Knocking out the growth hormone receptor (GHR) gene in mice produced a similar effect, extending the life span and concurrently exhibiting resistance to multiple forms of stress (Salmon et al, 2005). These long-lived mutants highlight the importance of the IGF-1 signaling pathway in modulating life span and in the coordinated modification of stress resistance. Seemingly utilizing different mechanisms, these long-lived rodents converge to a common stress-resistance phenotype. These facts implicate stress resistance as a surrogate for (and perhaps as a mediator of) longevity (Perez-Campo et al, 1998; Lithgow and Walker, 2002)

Methods

Results

Conclusion