Characterization of Mammalian In Vivo Enhancers Using Mouse Transgenesis and CRISPR Genome Editing [Book Chapter]

Book · 16 December 2021

DOI:https://doi.org/10.1007/978-1-0716-1847-9_11· OSTI ID:1907584

Osterwalder, Marco ^[1]; Tran, Stella ^[2]; Hunter, Riana D ^[2]; Meky, Eman M ^[2]; von Maydell, Kianna ^[2]; Harrington, Anne N ^[2]; Godoy, Janeth ^[2]; Novak, Catherine S ^[2]; Plajzer-Frick, Ingrid ^[2]; Zhu, Yiwen ^[2]; Akiyama, Jennifer A ^[2]; Afzal, Veena ^[2]; Kvon, Evgeny Z ^[3]; Pennacchio, Len A ^[4]; Dickel, Diane E ^[2]; Visel, Axel ^[5]

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1. Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of Bern (Switzerland)
2. Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
3. Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Univ. of California, Irvine, CA (United States)
4. Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); USDOE Joint Genome Institute (JGI), Berkeley, CA (United States); University of California, Berkeley, CA (United States)
5. Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); USDOE Joint Genome Institute (JGI), Berkeley, CA (United States); University of California, Merced, CA (United States

Embryonic morphogenesis is strictly dependent on tight spatiotemporal control of developmental gene expression, which is typically achieved through the concerted activity of multiple enhancers 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 enhancers and gene promoters. While epigenomic profiling and three-dimensional chromatin conformation capture have significantly increased the accuracy of identifying enhancers, assessment of subregional enhancer activities via transgenic reporter assays in mice remains the gold standard for assigning enhancer activity in vivo. Once this activity is defined, the ideal method to explore the functional necessity of a transcriptional enhancer and its contribution to target gene dosage and morphological or physiological processes is deletion of the enhancer sequence from the mouse genome. Here we present detailed protocols for efficient introduction of enhancer-reporter transgenes and CRISPR-mediated genomic deletions into the mouse genome, including a step-by-step guide for pronuclear microinjection of fertilized mouse eggs. We provide instructions for the assembly and genomic integration of enhancer-reporter cassettes that have been used for validation of thousands of putative enhancer sequences accessible through the VISTA 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 enhancer activities and sequence variants in mice, which is essential to understand mammalian genome function and genetic diseases.

• Research Organization: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• DOE Contract Number: AC02-05CH11231
• OSTI ID: 1907584
• Resource Relation: Related Information: Chapter 11 of Craniofacial Development Methods and Protocols
• Country of Publication: United States
• Language: English

Related Subjects

gene transcription
enhancers
cis-regulatory elements
pronuclear injection
transgenesis
transgenic mice
CRISPR
deletions
LacZ reporter
H11