The mutagen ENU can transfer its ethyl group to oxygen or nitrogen radicals present in DNA, which results in lesions that can cause mispairing during replication and eventually give rise to a single base pair substitution [23
]. We hypothesized that a deficiency in the repair system that detects or corrects such single base pair damages or mismatches would result in an increased ENU-induced mutation frequency and associated improvement of the target-selected mutagenesis-based knockout procedure. Indeed, deficiency of Msh6 was found to improve the ENU-driven knockout procedure in two ways; 1) it increases the ENU-induced germ line mutation frequency up to 1 mutation every 585 kb and 2) the mutation spectrum is changed and enhances the chance of generating a knockout-type allele by 21%. Cumulatively, this results in a ~2.5-fold increase in knockout efficiency.
The increased mutation frequency was reached at an ENU dose that was 25% lower than in wild types and together with the change in mutation spectrum this argues for the specificity of MMR-deficiency underlying the observed effects. In line with this, an increase in mutagenicity of ENU in MMR-deficient background was also shown in mouse ES cells lacking Msh2 [17
]. However, it has to be noted that in zebrafish, which lack Msh6, no difference in ENU-induced mutation frequency has been observed compared to MMR-proficient fish [26
]. This apparent contradiction with the results presented here could be explained by the large difference in mutation frequency. In wild type zebrafish, the ENU-induced mutation frequency is about 1 mutation every 100,000 bp, whereas, this frequency in wild type rats is more than 10-fold lower [5
]. Proposedly, the zebrafish mutation load is the maximum that is compatible with viability, a suggestion that is corroborated by comparable maximal mutation frequencies observed in C. elegans
] and Arabidopsis [14
]. Our results suggest that the lower ENU-induced mutation frequencies in rodents can at least partially be explained by more efficient mismatch repair in the testis that counteracts mutagenicity, and is less likely due to increased sensitivity to genotoxic damage in general. The decline of the mutation frequency in time that is observed in this study, however, could indicate the presence of genotoxic effect. Initial depletion of spermatogonial stem cells could be due to apoptosis induced by ENU damage – a mechanism that is presumed to be underlying the sterility effect at higher doses. Selective repopulation of the testis by the most viable stem cells with presumably the lowest amount of genotoxic damage, decreases the apparent mutation frequency (and potentially increases the change for clonal progeny). Our results indicate that the target-selected mutagenesis works most efficient for F1 progeny resulting from matings at about 10 – 14 weeks after the last ENU injection. However, it should be kept in mind that only F1 animals, generated after one full cycle of spermatogenesis (in rat and mouse about 60 to 70 days) after mutagenesis, should be screened in order to prevent retrieval of chimaeras. Such chimaeras can be induced by ethyl-DNA adducts in the fertilized oocyte that originate from mutagenized post-meiotic sperm cells and which could result in fixation of mutation in different lineages during embryonic development.
Besides the ENU mutagenesis approach, other gene targeting technologies are being developed for the rat, which include nuclear transfer, although to date no genetic modification has been reported, and knockdown by RNA interference (reviewed in [27
]). Recently, transposon-tagged mutagenesis [28
] was successfully applied to the rat [29
] as well and although this technique is currently less amendable for scaling, it should be considered as highly complementary to the existing ENU-based efforts. The ENU-driven target-selected mutagenesis approach has already been used successfully for generating a variety of novel rat knockout models [5
] and with the improvements described here, this approach does provide realistic technological requirements for screens on a genome-wide scale. In a wild type background, ~110,000 F1 rats would be needed to knockout any given gene with 95% probability (Figure ). This number is reduced to 40,000 in the MMR-deficient background. When reasoning the other way around, 40,000 F1 rats will contain knockouts of 95% of all rat genes and when considering 'only' 5,000 F1 rats, knockout alleles for ~50% of all the rat genes will be present. It should be said, however, that to identify these knockout alleles, the complete ORFeome would have to be screened. Although existing technologies are not suited for this, emerging massively parallel sequencing technologies [30
], and microarray-based enrichment procedures [31
] provide promising avenues in this direction. Keeping this in mind, archiving frozen sperm samples of mutant F1 animals, which can be recovered by intracytoplasmic sperm injection (ICSI) [33
] becomes very attractive.
Figure 3 Probability of gene knockouts in Msh6-deficient and wild type rats. The chance to retrieve a knockout for any given gene and the total number of genes that will be knocked out when all genes would be screened for mutations is plotted as a function of (more ...)
The identification of a wide range of potentially interesting missense mutations as well as the retrieval of four novel candidate knockout models resulting from the introduction of premature stopcodons by screening only a small set of about 300 animals illustrates the power of the approach. Furthermore, the ENU-based approach has the potential to generate allelic series (multiple mutations in the same gene) in different animals, which can facilitate the identification of novel gene functions. For example, hyper- and hypomorphic mutations provide information related to gene dosage effects and residues important for specific protein interactions or enzymatic functions can be identified.
Currently, the 4 knockouts and 45 selected missense mutations are being crossed to the next generation and bred to homozygosity for subsequent phenotypic analysis. As homozygous Msh6-deficiency, which could occur in later generations, would result in further accumulation of novel mutations, this outcross is also used to eliminate the mutant msh6
allele from the genetic background by genotype-assisted breeding. In addition, further outcrossing to wild type background should be performed and littermates should be included as control animals in phenotypic characterization experiments to minimize confounding effects of background mutations. Although such effects should never be ignored, estimates do indicate that this potential problem should not be exaggerated [35
], especially if outcrossing is combined with marker-assisted selection for which the outbred Wistar background is well-suited.