The Reciprocal Hemizygosity Test: A Novel Approach to Genotype-Phenotype Mapping
Genotype-phenotype mapping, identifying gene sequences and variations that are linked to phenotypic traits, remains a fundamental goal of genetic studies. Better understanding the genetic basis of traits, such as genetic disorders or disease susceptibility and resistance, can allow for more effective methods of disease prevention, diagnosis, management, and therapy. Most current methods of genotype-phenotype mapping are only able to investigate trait variation in interbreeding populations. A novel approach called the reciprocal hemizygosity test has been recognized for extending the study to related but reproductively isolated species, which have differing phenotypic traits fixed after long evolutionary divergence. The test involves generating and mutagenizing hemizygotes from the divergent species. Mutagenized cells are then tested for gain or loss of the phenotype under study, and high-throughput sequencing is performed to identify responsible sequences. However, despite its potential, the test has only been performed in lower organisms and is yet to be tested in higher eukaryotes.
This study applies the reciprocal hemizygosity test to investigate the genetic basis for enhanced CNS axon regeneration in the wild-caught CAST/EiJ mouse strain compared to standard laboratory mouse strains. Random viral mutagenesis of a hybrid embryonic stem cell line derived from a cross between the CAST/EiJ and 129 strains produced a pool of mutagenized hemizygotes. Using directed differentiation, motor neurons were generated from parent strains and the hemizygotes, characterized through immunohistochemical analysis. A microfluidic device was designed to provide the controlled microenvironment to select cells with enhanced neuronal regeneration capability. In future experiments, genome sequencing of neurons selected for enhanced regeneration will identify candidate genes potentially responsible for the phenotype in CAST/EiJ mice. Using the axon regeneration trait, this project serves as an example for applying the reciprocal hemizygosity test in metazoans.