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. 2016 Jul 21;11(7):e0159377.
doi: 10.1371/journal.pone.0159377. eCollection 2016.

Improvement of ENU Mutagenesis Efficiency Using Serial Injection and Mismatch Repair Deficiency Mice

Jabier Gallego-Llamas  1   2 , Andrew E Timms  1 , Rose Pitstick  3 , Janet Peters  3 , George A Carlson  3 , David R Beier  1   2
Affiliations

Improvement of ENU Mutagenesis Efficiency Using Serial Injection and Mismatch Repair Deficiency Mice

Jabier Gallego-Llamas et al. PLoS One. .

Abstract

ENU mutagenesis is a powerful method for generating novel lines of mice that are informative with respect to both fundamental biological processes and human disease. Rapid developments in genomic technology have made the task of identifying causal mutations by positional cloning remarkably efficient. One limitation of this approach remains the mutation frequency achievable using standard treatment protocols, which currently generate approximately 1-2 sequence changes per megabase when optimized. In this study we used two strategies to attempt to increase the number of mutations induced by ENU treatment. One approach employed mice carrying a mutation in the DNA repair enzyme Msh6. The second strategy involved injection of ENU to successive generations of mice. To evaluate the number of ENU-induced mutations, single mice or pooled samples were analyzed using whole exome sequencing. The results showed that there is considerable variability in the induced mutation frequency using these approaches, but an overall increase in ENU-induced variants from one generation to another was observed. The analysis of the mice deficient for Msh6 also showed an increase in the ENU-induced variants compared to the wild-type ENU-treated mice. However, in both cases the increase in ENU-induced mutation frequency was modest.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Breeding scheme for analysis of serial ENU-treatment in wild-type and MMR defective mice.
G01 males carry a random set of de novo point mutations induced by ENU treatment of wild-type or MMR-mutant G0 mice. G02, G03 and G04 carry mutations that were induced in their respective parents, as well as those inherited from previous generations. Each generation treated with ENU will on average have 3000–6000 new ENU-induced variants genome wide. After ENU treatment each male is crossed with a wild-type female; their progeny will inherit newly induced mutations and 50% of those of the parent. The mutations are sampled by exome analysis; the expected number of ascertained mutations (E) is shown. The ENU treatment was performed on three successive generations for the Msh6+/+ mice and four generation for the Msh6+/- mice. Msh6-/- mice did not tolerate ENU treatment.
Fig 2
Fig 2. Effect of ENU treatment on survival.
Msh6+/- males show survival comparable to wild-type males after treatment with the standard concentration of ENU (90mg/kg x 3). Survival of ENU-treated Msh6-/-males is much reduced. Death is expressed in days.
Fig 3
Fig 3. ENU-induced mutation spectrum.
(a) Frequencies of mutations in successive generation of Msh6+/+ mice in the pooled samples. (b) Frequencies of mutations in the successive generation of Msh6+/- mice in the pooled samples.
See this image and copyright information in PMC

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