UC Irvine

Embryonic morphogenesis is strictly dependent on tight spatiotemporal control of developmental gene expressionGene expression, which is typically achieved through the concerted activity of multiple enhancersEnhancers driving cell type-specific expression of a target gene. Mammalian genomes are organized in topologically associated domains, providing a preferred environment and framework for interactions between transcriptional enhancersEnhancers and gene promotersPromoters. While epigenomic profiling and three-dimensional chromatin conformation capture have significantly increased the accuracy of identifying enhancersEnhancers, assessment of subregional enhancerEnhancers activities via transgenic reporter assays in miceMice remains the gold standard for assigning enhancerEnhancers activity in vivo. Once this activity is defined, the ideal method to explore the functional necessity of a transcriptional enhancerEnhancers and its contribution to target gene dosage and morphological or physiological processes is deletion of the enhancerEnhancers sequence from the mouseMice genome. Here we present detailed protocols for efficient introduction of enhancer-reporter transgenes and CRISPR-mediated genomic deletionsGenomic deletions into the mouseMice genome, including a step-by-step guide for pronuclear microinjection of fertilized mouseMice eggs. We provide instructions for the assembly and genomic integration of enhancer-reporter cassettes that have been used for validation of thousands of putative enhancerEnhancers sequences accessible through the VISTA enhancer browserVISTA Enhancer browser, including a recently published method for robust site-directed transgenesis at the H11 safe-harbor locus. Together, these methods enable rapid and large-scale assessment of enhancerEnhancers activities and sequence variants in miceMice, which is essential to understand mammalian genome function and genetic diseases.