Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo
Open Access
- 28 September 2021
- journal article
- research article
- Published by Springer Science and Business Media LLC in BMC Research Notes
- Vol. 14 (1), 1-8
- https://doi.org/10.1186/s13104-021-05785-4
Abstract
The mammalian Notch ligand DLL1 has essential functions during development. To visualise DLL1 in tissues, for sorting and enrichment of DLL1-expressing cells, and to efficiently purify DLL1 protein complexes we tagged DLL1 in mice with AcGFPHA or Strep/FLAG. We generated constructs to express DLL1 that carried C-terminal in-frame an AcGFPHA tag flanked by loxP sites followed by a Strep/FLAG (SF) tag out of frame. Cre-mediated recombination replaced AcGFP-HA by SF. The AcGFPHAstopSF cassette was added to DLL1 for tests in cultured cells and introduced into endogenous DLL1 in mice by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cells and embryo lysates. In CHO cells the AcGFP fluorophore fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the SF tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or SF-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.Keywords
Funding Information
- Tistou & Charlotte Kerstan Stiftung
- DFG Excellence Cluster Rebirth
- Medizinischen Hochschule Hannover
- Medizinische Hochschule Hannover (MHH)
This publication has 38 references indexed in Scilit:
- Putting into Practice Domain-Linear Motif Interaction Predictions for Exploration of Protein NetworksPLOS ONE, 2011
- Dll1- and Dll4-Mediated Notch Signaling Are Required for Homeostasis of Intestinal Stem CellsGastroenterology, 2011
- Strep/FLAG Tandem Affinity Purification (SF‐TAP) to Study Protein InteractionsCurrent Protocols in Protein Science, 2009
- The intracellular region of Notch ligands Dll1 and Dll3 regulates their trafficking and signaling activityProceedings of the National Academy of Sciences of the United States of America, 2008
- Widespread Delta-Like-1 Expression in Normal Adult Mouse Tissue and Injured Endothelium Is Reflected by Expression of the Dll1LacZ LocusJournal of Vascular Research, 2007
- Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivoThe Journal of cell biology, 2007
- A Comparative Analysis of Novel Fluorescent Proteins as Reporters for Gene Transfer StudiesJournal of Histochemistry & Cytochemistry, 2007
- Premature myogenic differentiation and depletion of progenitor cells cause severe muscle hypotrophy in Delta1 mutantsProceedings of the National Academy of Sciences of the United States of America, 2007
- Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivoNature Immunology, 2004
- mDll1 and mDll3 expression in the developing mouse brain: Role in the establishment of the early cortexJournal of Neuroscience Research, 2001