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 paraquat-resistant (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.

Fig. 1

ES cell mutagenesis and selection for resistance to oxidative stress. ES cells mutagenized by ENU were plated into ten 96-well plates and a set of replica plates were prepared for “sib-selection,” while the original “masters” were frozen. Cells on the replica were treated with PQ (40 μM) to identify wells with resistant cells. Cells in the corresponding well on the master plates were then thawed and plated at low density onto culture dishes. Individual ES cell colonies were then hand-picked to a 96-well plate for a second round of sib-selection (one 96-well plate was sufficient). The resistant ES cells thus identified from the second master plate were clonally derived and free of exposure to stress or selection agents

Fig. 2

Characterization of oxidant-resistant ES cells. a Number of resistant ES cell colonies under various PQ doses. Resistant clones (1D3, 1E1, 2D12, 3B7, 4C11, 5A4) exhibit PQ resistance to various extents compared with either the parental ES cells (c7.1) or an arbitrarily selected ENU-mutagenized ES cell clone (Arbit). Each data point represents a mean ± SD (n = 3). b Total reactive oxygen species (ROS) production in ES cells during oxidative-stress treatment. Upon PQ treatment, parental ES cells (c7.1) showed increased ROS in a dose-dependent manner. Resistant ES cell clones 4C11 and 5A4, on the other hand, showed a less pronounced increase in ROS upon PQ treatment. The percentage increase in ROS in 4C11 and 5A4 ES cells treated with 500 μM or 1 mM PQ was significantly lower than that of the parental ES cells. Error bars represent SD of means (n = 5). Significance of differences in ROS generation between parental and stress-resistant ES cells was analyzed by one-way ANOVA

Fig. 3

Characterization of oxidant-resistant ES cells. a Morphology of ES cell colonies in culture. Parental ES cell line c7.1 and two resistant lines (1E1 and 4C11) are germline competent and also exhibit a compacted colony morphology with a tight boundary, a characteristic typical of pluripotent ES cells. Clone 5A4, however, does not retain this morphology and, indeed, this line failed to generate mice. b Fixed ES cell colonies stained with OCT4 antibody. Compared with germline-competent clones (c7.1, 1E1, and 4C11), 5A4 appears to have lower levels of the OCT4 protein. This reduced OCT4 level may correlate with the inability of the 5A4 clone to produce chimeric mice

Fig. 4

Assessment of stress resistance in skin-derived fibroblasts. Paraquat resistance of fibroblasts isolated from the tail skin of chimeric mice derived from injection of 1E1 and 4C11 ES cells. Fibroblasts (passage 3) were treated with PQ for 6 h in culture. After 20 h of recovery, cell viability was measured by the WST-1 cell proliferation assay. Untreated fibroblasts were normalized to 100% viability. a Fibroblasts from the tail of chimera (928) and an F₁ male (1005) offspring were more resistant to PQ than either B6/129 F₁ hybrid mice controls or five littermates (Littermate, only one is shown). b Fibroblasts from two chimera (6291 and 6293) and an F₁ male mouse (4008) were resistant to PQ compared with fibroblasts from B6/129 hybrid mice and two littermates (Littermate, only one was shown). * P<0.0001 (survival measured at 2 mM PQ) between a group of six B6/129 control mice and the resistant mouse, evaluated by one-way ANOVA. Error bars represent SD of means (n = 3). c ROS production in fibroblasts during oxidant challenge. ROS levels in fibroblasts from F₁ mice (1005 and 4008) treated with 100, 500, and 1000 μM of PQ were significantly less than that in fibroblasts isolated from wild-type controls (B6/129, only one is shown), as evaluated by one-way ANOVA. Error bars represent SD of means (n = 5)