Genetic suppression of cryoprotectant toxicity

Abstract

We report here a new, unbiased forward genetic method that uses transposon-mediated mutagenesis to enable the identification of mutations that confer cryoprotectant toxicity resistance (CTR). Our method is to select for resistance to the toxic effects of M22, a much-studied whole-organ vitrification solution. We report finding and characterizing six mutants that are resistant to M22. These mutants fall into six independent biochemical pathways not previously linked to cryoprotectant toxicity (CT). The genes associated with the mutations were Gm14005, Myh9, Nrg2, Pura, Fgd2, Pim1, Opa1, Hes1, Hsbp1, and Ywhag. The mechanisms of action of the mutations remain unknown, but two of the mutants involve MYC signaling, which was previously implicated in CT. Several of the mutants may up-regulate cellular stress defense pathways. Several of the M22-resistant mutants were also resistant to dimethyl sulfoxide (Me₂SO), and many of the mutants showed significantly improved survival after freezing and thawing in 10% (v/v) Me₂SO. This new approach to overcoming CT has many advantages over alternative methods such as transcriptomic profiling. Our method directly identifies specific genetic loci that unequivocally affect CT. More generally, our results provide the first direct evidence that CT can be reduced in mammalian cells by specific molecular interventions. Thus, this approach introduces remarkable new opportunities for pharmacological blockade of CT.

Keywords: Cryoprotectant toxicity resistance; Cryoprotective mutants; Forward genetics; Freezing injury; Mice; Mutant selection; Stem cells; Stress resistance; Transposons.

Fig. 1.

Overview of strategy for identifying and studying CTR mutants. Mutants are generated and (1) selected for CTR. (2) Mutants are confirmed or rejected. (3) Mutations putatively conferring CTR are identified. (4) Causality is verified by demonstrating that CTR is conferred by other mutations in the candidate gene and is abolished by excising the pB transposon and (5) generating genomic equivalents of that mutation. (6) Bioinformatics are used to link the identified mutation to previously-identified pathways. (7) A search is made to identify known drugs that may induce CTR. Candidate drugs are then tested on wild-type cells for efficacy in CTR. Drug effects are characterized (8) molecularly and (9) phenotypically. (10) Ultimately, mice carrying the original mutation are generated and novel improved drugs are developed using a variety of distinct methods, and (11) their organs are tested for CTR.

Fig. 2.

Independent counts of cell colonies by different scorers.

Fig. 3.

Survival of mouse C9 cells during exposure to 60% M22 at 2°C, by the method of Guan et al. [36]. Shown are the pooled results of two experiments.

Fig. 4.

A. Morphology of C9 cells under various M22 conditions, at 37°C. Images obtained at 0%, 6%, and 9% M22 after 48 and 96 hours of exposure. Note decreasing size and increasingly smooth border of colonies or cells as M22 concentration increases. B. Dose-response effects of exposing C9 cells to M22 for 96 hours at 37°C.

Fig. 5.

A. Survival (measured by the MTT assay) of controls (circles), mutant M2.1/Gm14005 (squares), and mutant M4.3/Myh9 (diamonds), with 0 – 6% M22 at 2°C for 48 hours. Mutant measurements are normalized to controls. All p-values are independent, one-tailed t-tests of means: *, p<0.05; †, p< 0.01; ‡, p<0.001. B. Survival (measured by the MTT assay) of mutants M2.1/Gm14005 and M4.3/Myh9 exposed to 0 – 5% (w/v) Me₂SO at 37°C for 48 hours. Symbols represent mutants and significance levels as in A.

Fig. 6.

Mutant viability after 5-minute exposure to 10% (v/v) Me₂SO at 37°C, followed by freezing at −80°C, storage for 24 hours, and thawing. Mutants represented include M2.1/Gm14005 (squares), M2.2/Nrg2/Pura (triangles), M3.1/Fgd2/Pim1 (“X”), M4.2/Opa1/Hes1 (“+”), M4.3/Myh9 (diamonds), and M5.1/ Hsbp1/Ywhag (horizontal bars). A-C: Trypan blue assay: Mutant proportions alive are normalized to control proportion alive. In the first experiment shown (panel 6A) mutant M2.1/Gm14005 displayed resistance 12.7-fold more than controls; that exceptional value is annotated within the graph. All other fold-values greater than controls were less than 5. Controls are represented by circles. All p-values are tests of independent proportions alive: *, p<0.05; †, p< 0.01; ‡, p<0.001. D-F: Colony formation assay: Counts of mutant colonies are normalized to control counts. All p-values are independent, one-tailed t-tests of means. Symbols representing mutants and significance levels are as in A-C.