A gene-driven approach to the identification of ENU mutants in the mouse

There is increasing awareness of the need for a comprehensive functional annotation of the mouse genome¹. Phenotype-driven approaches to mouse mutagenesis using the chemical mutagen ENU are useful for generating large numbers of new mutants for systematic studies of mammalian gene function^{2, 3}. In principle, 355GT ENU mutagenesis programs also provide a route to undertake gene-driven approaches. One approach to gene-driven mutagenesis is to establish parallel DNA and sperm archives of ENU-mutagenized mice. Rapid screening of DNA sequences from the archive is followed by recovery of identified mutants from the sperm bank. The advantage of this approach is that it allows recovery of the full range of possible functional changes in a gene, including hypomorphs, antimorphs and neomorphs.

Figure 1. Probabilities of finding mutant alleles of a gene in DNA sequence from F1 offspring of ENU-mutagenized male mice. The graphs show the probabilities of finding n or more mutant alleles of a gene of interest in varying numbers of F1 DNA sequences analyzed, calculated with BINOM v. 1.72 (J. Ott) using a mutation rate of 0.00108 mutations per locus. Probabilities were summed to arrive at the approximate probability of n or more alleles being found. Full Figure and legend (53K)

We screened the complete DNA archive for four genes and identified many silent, missense and stop mutations (Table 1). Two missense and one stop mutation were identified in Gjb2. Mutations in human GJB2 are the most common cause of nonsyndromic deafness in the human population^{6, 7, 8, 9}, including recessive and dominant alleles^{6, 10}. In contrast, a mouse knock-out of Gjb2 is homozygous-lethal owing to placental defects¹¹. We did not detect the stop mutation (355GT, Glu119Stop) in any of the other progeny screened, including sibs and other progeny of the mutagenized male from which the mutant mouse was derived. We recovered the mutant mouse from the frozen sperm archive using in vitro fertilization of C3H eggs¹². Twenty-six animals were born, of which 10 were wildtype and 16 carried the mutation. All of the mutant mice were screened for deafness using a calibrated click box that emits high frequency sound¹³. At six weeks, all of the heterozygous mutant mice showed a Preyer reflex indicative of a normal hearing response. We also intercrossed heterozygous mutant mice to examine the phenotype of homozygotes. Of 61 progeny recovered, 37 were heterozygous and 24 wildtype. No homozygous mutants were identified, indicating that the mutation is embryonic-lethal. Complementation studies intercrossing mice heterozygous with respect to the Glu119Stop mutation with Gjb2 knockout heterozygous mice failed to produce any compound heterozygotes carrying both mutations (of 51 progeny analyzed), confirming that lethality is due to the Glu119Stop mutation.

Table 1. Identification of mutations from the ENU DNA archive Full Table

It has been estimated that the overall level of sequence change (including silent and functional changes) induced by ENU could be as high as 1 in 100 kb (ref. 14). Empirical data is sparse, however—though preliminary screening of 370 kb of a small cohort of mice revealed up to six sequence changes¹⁴. In the UK ENU program, mutagenized BALB/c males are crossed to C3H females that carry the recessive retinal degeneration rd1 mutation at the Pde6b locus, allowing us to estimate a specific locus mutation rate for this gene. In 6,500 mice screened, we identified seven alleles at the rd1 locus, giving a mutation rate of 1.08 10^-3 (C. Thaung et al., unpublished data). Given that the coding sequence of Pde6b is 2.568 kb, we might expect, extrapolating genome-wide, a functional change for every 2.38 Mb of coding sequence screened. This equates to the numbers of functional changes identified in the screening of the DNA archive, from which, of a total of 9.48 Mb, we recovered two missense and one stop mutation. The absence of functional changes in the other genes screened underlines the stochastic nature of the screen (although we identified two silent mutations in two genes), which can be addressed through bigger archives (Fig. 1). It is also likely that mutations were missed by screening with denaturing high-performance liquid chromatography; this may explain the apparently low level of silent mutations detected. Improvements in mutation scanning of genomic sequence, both in sensitivity and rapidity, will aid this process. We show here, however, that gene-driven screens using parallel DNA and sperm archives will facilitate the generation and recovery of point mutations in mouse genes and the functional annotation of the mouse genome.

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Competing interests statement:  The authors declare that they have no competing financial interests.