Characterization of Mammalian In Vivo Enhancers Using Mouse Transgenesis and CRISPR Genome Editing.

Osterwalder, Marco; Tran, Stella; Hunter, Riana D; Meky, Eman M; von Maydell, Kianna; Harrington, Anne N; Godoy, Janeth; Novak, Catherine S; Plajzer-Frick, Ingrid; Zhu, Yiwen; Akiyama, Jennifer A; Afzal, Veena; Kvon, Evgeny Z; Pennacchio, Len A; Dickel, Diane E; Visel, Axel (2022). Characterization of Mammalian In Vivo Enhancers Using Mouse Transgenesis and CRISPR Genome Editing. Methods in molecular biology, 2403, pp. 147-186. Springer 10.1007/978-1-0716-1847-9_11

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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.

Item Type:	Journal Article (Further Contribution)
Division/Institute:	04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR)
UniBE Contributor:	Osterwalder, Marco
Subjects:	600 Technology > 610 Medicine & health
ISSN:	1940-6029
Publisher:	Springer
Language:	English
Submitter:	Jasmine Stiefel
Date Deposited:	14 Dec 2022 07:34
Last Modified:	14 Dec 2022 18:39
Publisher DOI:	10.1007/978-1-0716-1847-9_11
PubMed ID:	34913122
Uncontrolled Keywords:	CRISPR Deletions Enhancers Gene transcription H11 LacZ reporter Pronuclear injection Transgenesis Transgenic mice cis-regulatory elements
URI:	https://boris.unibe.ch/id/eprint/175819