Treatment of animals with a mutagenic compound that introduces random mutations in the germ line is a very fast and efficient method for introducing a wide range of mutations in large sets of genes in vivo
. In rodents, ENU has been shown to be the most potent chemical germ line mutagen.1
ENU treatment of male animals causes adducts in the DNA of spermatogonial stem cells, which after several rounds of cell division, result in random point mutations and mutagenized sperm.2
F1 animals derived from outcrosses with wild-type females carry random heterozygous ENU-induced mutations in their genome. Subsequently, the DNA of these animals can be screened by a variety of techniques for the presence of mutations in pre-selected genes of interest,3, 4
with the goal to identify animals that carry induced variants that affect normal protein function, for example, by the introduction of a premature stop or by affecting functionally important residues.
The laboratory rat Rattus norvegicus
is one of the most used model organisms in biomedical research and has been the preferred model for studying human physiology and pathology.5
As a highly diverged mammalian model (~60 million years with human and 20–40 million years with mouse6
), the rat is highly complementary to the mouse, enabling phenotypic comparison of gene knockouts in both mammals to better understand the specific gene function in human biology. In addition, in specific cases the rat can have advantages in studying mammalian physiology and biology due to its relative large body size and the availability of well-established behavioral and neurological assays.7
Although most rat knockout models have thus far been generated through ENU-driven approaches, only recently alternative technologies emerged. Transposon-tagged mutagenesis,8
zinc-finger nuclease-mediated knockout generation9
and the isolation of pluripotent ES cells that potentially can be used for gene targeting10, 11
now provide a range of possibilities for manipulating the rat genome and promises to boost the use of the rat as a versatile genetic model system. ENU-driven target-selected mutagenesis has specific characteristics that make it an attractive technology that is complementary to the other approaches.12
First, it is a relatively simple technology without any cell or oocyte manipulation steps. Second, it can easily be scaled up for high throughput and is a relatively cheap method, especially in terms of the number of animals used per knockout (in this paper ~100 rats). Third, it offers the possibility to identify (allelic series of) more subtle variation because of amino acid changes that result in hyper- and hypomorphic alleles.3, 4
One of the major disadvantages of the ENU-based approach was its relative inefficiency. However, recently we increased the efficiency by about 2.5-fold by taking advantage of DNA mismatch repair (MMR)-deficiency in the MSH6 knockout rat,13, 14
a system known to be involved in repairing ENU-induced lesions in the genome.15
Further efficiency improvements can be expected by implementing next-generation sequencing technology for mutation discovery. Another drawback of the method is that mutation generation is random and that only the discovery is done in a targeted fashion. In other words, generation of knockouts is relatively efficient, but obtaining a knockout for a specific gene is still challenging. However, ENU-driven target-selected mutagenesis is a versatile technology for the systematic generation of large catalogs of knockouts and allelic variants of gene families or eventually all protein-coding genes. The latter approach in combination with efficient cryopreservation and rederivation protocols would generate a unique genome-wide resource for knockouts as well as mutant alleles reflecting human genetic variation.
Here, we applied the improved ENU-driven target-selected mutagenesis method for generating a unique resource of in vivo
GPCR mutant rat models consisting of both knockouts as well as (allelic series of) missense mutations. G-protein-coupled receptors (GPCRs) are 7 transmembrane (TM) receptors, which regulate many cellular processes, including the senses of taste, smell, and vision and control a myriad of intracellular signaling systems in response to external stimuli. Importantly, many diseases are linked to GPCRs and they represent by far the largest class of targets for current drugs as well as for the development of novel small-molecule medicines.16
Moreover, because of their role in the regulation of cellular function they are arguably one of the best-studied classes of proteins, although for many GPCRs their ligand as well as biological function remains to be elucidated. Furthermore, genetically altered GPCR animal models are scarce, especially in non-murine species, The use of a random mutagenesis approach for the generation of GPCR mutants is in principle very well suited for understanding the in vivo
receptor function as new insights can be obtained by completely knocking out specific receptors, but also by changing functionally important residues, for example, involved in ligand binding or second messenger signal transduction. Importantly, the high structural conservation between the different GPCRs allows for confident prediction of possible effects of amino acids changes. We systematically applied the ENU-driven target-selected mutagenesis approach to a set of about 250 rat GPCRs that have clear orthologs to human GPCRs. In total, we identified 131 non-synonymous mutations in 99 different GPCRs, including 7 novel potential knockout alleles and 45 missense mutants that were predicted to affect specific GPCR function or stability of folding of the protein. Characterization of selected models shows that ENU target-selected mutagenesis is a powerful and efficient approach for in vivo
functional studies on G-protein-coupled receptors.