Transmission of mutant phenotypes from ES cells to adult mice

Abstract

Genetic manipulation of embryonic stem (ES) cells has been used to produce genetically engineered mice modeling human disorders. Here we describe a novel, additional application: selection for a phenotype of interest and subsequent transmission of that phenotype to a living mouse. We show, for the first time, that a cellular phenotype induced by ENU mutagenesis in ES cells can be transmitted and recapitulated in adult mice derived from these cells. We selected for paraquatresistant (PQ R ) ES clones. Subsequent injection of these cells into blastocysts resulted in the production of germline chimeras, from which tail skin fibroblasts exhibited enhanced PQ R . This trait was also recovered in progeny of the chimera. We avoided PQ toxicity, which blocks the ability to involve the germline, by developing a sib-selection method, one that could be widely applied wherever the selection itself might diminish the pluripotency of the ES cells. Thus, phenotype-driven screens in ES cells are both feasible and efficient in producing intact mouse models for in vivo studies.

25years after age-1: Genes, interventions and the revolution in aging research

“…Further communications will review other work from the Johnson lab as we began studies of the role of stress in the specification of lifespan (Lithgow et al, 1995; Melov et al, 1995; Johnson et al, 1996; Murakami and Johnson, 1998; Henderson and Johnson, 2001), hormesis (Cypser and Johnson, 2003), dietary restriction (Park et al, 2009), and stochastic effects (Rea et al, 2005), all in C. elegans , as well as our very recent work on dietary restriction in the mouse (Liao et al, 2010) and the selection of mutants in the mouse, using embryonic stem cells (Chick et al, 2009). …”

“…We developed two strategies for identifying Age mutants in C. elegans (de Castro et al, 2004; Duhon et al, 1996), but neither was widely applied due to the laborious nature of their application. (However, the use of stress resistance as a surrogate marker coupled with transposon-tagging has recently been used to identify putative longevity mutants directly in mouse embryonic stem cells (Chick et al, 2009; Chick and Johnson, unpublished).…”

Generation of pig primary fibroblast cells harboring defective <i>MC</i>4R genes by <i>N</i>-ethyl-<i>N</i>-nitrosourea mutagenesis: A gene-driven, nontransgenic approach to pig improvement

“…Greber et al [5] treated E14.1 ES cells with 1.5 mM ENU for 90 min; one out of two mutant cell clones gave rise to germ-line chimeras. Chick et al [4] mutagenized c7.1 ES cells with 4.3 mM ENU for 2 h, and chimera formation using two of three mutagenized cell clones resulted in germ-line transmission. These results suggest that pig fibroblast cell clones harboring S132P or I185S would give rise to cloned pigs upon SCNT, because the per-base mutation frequency introduced into these pig cells in the present study seems no higher than that introduced into the mutant ES cells mentioned above.…”

“…Mutant mice derived from embryonic stem (ES) cells mutagenized with chemical mutagens such as N-ethyl-N-nitrosourea (ENU) in vitro have been produced [3][4][5][6][7]. Somatic cell nuclear transfer (SCNT) of pigs has been developed, and many gene-knockout pigs have been generated by exploiting this technology [1,8].…”

Screening for stress-resistance mutations in the mouse

“…In these initial proof-of-principal studies, we used N-ethyl-N-nitrosourea (ENU) to generate a highly mutagenized ES cell population, which facilitated the isolation of stress-resistant mutants (Chick et al, 2009). Although the screen was successful, it suffered from two drawbacks.…”