Using i-GONAD for Cell-Type-Specific and Systematic Analysis of Developmental Transcription Factors In Vivo

SIMPLE SUMMARY: Gene activity is regulated by transcription factors and interacting proteins that bind to chromosomes in cells of developing embryos, adult organisms, and during disease. Thus, it is important to understand how transcription factors function in a specific biological context. By combining established approaches, this study outlines an efficient strategy to generate mouse models that facilitate the analysis of transcription factors in defined cell types. These mouse models improve existing methods for the identification of interacting proteins and chromosomal binding sites of transcription factors. Two transcription-factor-encoding genes with important functions in the developing nervous system and an association with neurodevelopmental disorders were genetically modified in mice and will serve as valuable tools for the investigation of nervous system development and related disease. ABSTRACT: Transcription factors (TFs) regulate gene expression via direct DNA binding together with cofactors and in chromatin remodeling complexes. Their function is thus regulated in a spatiotemporal and cell-type-specific manner. To analyze the functions of TFs in a cell-type-specific context, genome-wide DNA binding, as well as the identification of interacting proteins, is required. We used i-GONAD (improved genome editing via oviductal nucleic acids delivery) in mice to genetically modify TFs by adding fluorescent reporter and affinity tags that can be exploited for the imaging and enrichment of target cells as well as chromatin immunoprecipitation and pull-down assays. As proof-of-principle, we showed the functional genetic modification of the closely related developmental TFs, Bcl11a and Bcl11b, in defined cell types of newborn mice. i-GONAD is a highly efficient procedure for modifying TF-encoding genes via the integration of small insertions, such as reporter and affinity tags. The novel Bcl11a and Bcl11b mouse lines, described in this study, will be used to improve our understanding of the Bcl11 family’s function in neurodevelopment and associated disease.