Rat reverse genetics : generation and characterization of chemically induced rat mutants

Abstract

The use of animal models has been crucial for studying the function of genetic elements in the human genome. Embryonic stem (ES) cell-based homologous recombination (HR) has proven a very efficient technique for gene manipulation. However, this technique is not (yet) available for all model organisms due to the inability of maintaining a pluripotent state of ES cells in culture. The laboratory rat Rattus norvegicus is one of the most used model organisms in biomedical research, especially for studying physiology, pharmacology, toxicology and neurobiology. Due to the long lack of pluripotent rat ES cells, alternative methods to manipulate its genome have been developed. The first genetic rat knockout models were developed using chemically-induced mutagenesis, which is based on treating male rats with the mutagen N-ethyl-N-nitrosourea (ENU) that very efficiently introduces point mutations in the spermatogonial stem cells (SSCs). Subsequently, the ENU-treated rats are crossed with untreated rats to generate an F1 population in which each animal contains unique heterozygous mutation throughout its genome. Finally, mutations that affect gene function are identified and crossed to homozygocity in order to study its effect. The technique can be used for both forward (phenotype-driven) and reverse (gene-driven) genetics approaches. In this thesis the optimization and application of the reverse genetics procedure, termed target-selected mutagenesis, in the rat has been described. In this approach the DNA of F1 animals is screened for mutations in pre-selected genes using molecular biological techniques. The efficiency of the approach depends on 1) the mutation frequency and 2) mutation discovery. By taking advantage of DNA mismatch repair (MMR)-deficiency, a system involved in repairing ENU-induced DNA damage, in the MSH6 knockout rat, we demonstrate that the mutation frequency can be significantly increased. After obtaining a F1 library, different techniques have been applied to identify interesting mutations. Here, we combine massive parallel sequence technology with microarray-based enrichment of genomic regions of interest to further improve the mutation discovery efficiency. The increased efficiency of the approach was demonstrated by screening a large set of genes in the rat encoding one-to-one orthologs of human G protein-coupled receptors (GPCRs). This large family of seven transmembrane receptors play essential roles in physiology and disease; however, genetically altered GPCR animal models are scarce, especially in non-murine species. Using this approach multiple mutant rat lines were isolated, including knockout as well as missense animals. Extensive bioinformatic analyses were applied in order to prioritize the missense mutants. We show loss of function for a knockout allele of the melanocortin 4 receptor (Mc4r) and a missense allele of the lysophosphatidic acid receptor 1 (Lpar1) confirming our analyses. ENU mutagenesis is a non-redundant gene manipulation technique with several unique advantages. It is highly efficient and random in introducing germ line point mutations, which resemble the most common form of human genetic variation. Furthermore, it allows for allelic series production, which can be important for studying specific gene function. In conclusion, using chemically-induced mutagenesis we have significantly contributed to the generation and characterization of genetically manipulated rats that model human disease.