Hannah Schrader Dear, Nicole E Franks, Ella E Markley, Alexander M Holtz, Jane Y Song, Craig N Johnson, Paola I Medina-Cabrera, Daniela Hernandez, Praise Joel, Marina Pasca di Magliano, Deneen M Wellik, Benjamin L Allen
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引用次数: 0
Abstract
GLI proteins (GLI1-3) are the transcriptional effectors of mammalian Hedgehog (HH) signaling. However, studies of GLI function have been hampered by the lack of robust GLI antibodies. To address this, we utilized CRISPR-based gene editing to generate endogenous epitope-tagged Gli alleles for each Gli gene (Gli1FLAG, Gli2HA, Gli3V5). Through breeding, we established a novel mouse model, Gli1FLAG/FLAG;Gli2HA/HA;Gli3V5/V5, referred to as GliFHV. Importantly, GliFHV animals are viable and fertile with no overt phenotypes. Sanger and long-range DNA sequencing confirmed proper editing of each Gli allele, while qPCR and western blot analysis confirmed similar gene expression and protein levels, respectively, between wildtype and GliFHV animals. We utilized these mice to assess GLI localization in the developing limb, finding that all three GLIs localize to primary cilia with distinct distributions. Finally, we generated immortalized GliFHV mouse embryonic fibroblasts (MEFs), demonstrating that these cells are HH-responsive and that GLIs localize to primary cilia and nuclei in a HH-dependent fashion. These animals and cell lines provide a valuable resource for analyses of GLI processing, localization and function throughout embryogenesis, postnatal development, and in adults.
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Development’s scope covers all aspects of plant and animal development, including stem cell biology and regeneration. The single most important criterion for acceptance in Development is scientific excellence. Research papers (articles and reports) should therefore pose and test a significant hypothesis or address a significant question, and should provide novel perspectives that advance our understanding of development. We also encourage submission of papers that use computational methods or mathematical models to obtain significant new insights into developmental biology topics. Manuscripts that are descriptive in nature will be considered only when they lay important groundwork for a field and/or provide novel resources for understanding developmental processes of broad interest to the community.
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To aid navigability, Development has dedicated sections of the journal to stem cells & regeneration and to human development. The criteria for acceptance into these sections is identical to those outlined above. Authors and editors are encouraged to nominate appropriate manuscripts for inclusion in one of these sections.
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