Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo

ABSTRACT: Objective DLL1 is a mammalian Notch ligand with essential functions during embryonic development. We tagged endogenous DLL1 in one homologous recombination step such that AcGFP-HA tagged DLL1 could be converted by Cre-mediated site-specific recombination into StrepFlag tagged DLL1. We anticipated that this should allow us to visualise DLL1 in living cells as well as allow for sorting and enrichment of DLL1-expressing cells and efficient purification of DLL1 protein complex. Results We generated constructs to express a DLL1 variant that carried C-terminal in frame an AcGFPHA tag flanked by loxP sites followed by a StrepFlag tag out of frame. Cre-mediated recombination removed AcGFP-HA and added StrepFlag in frame to DLL1. The AcGFPHAstopStrepFlag tag was added to the Dll1 cDNA to allow for tests in cultured cells in vitro and was introduced into endogenous DLL1 in mice using ES cells modified by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cell and embryo lysates. In CHO cells the AcGFP protein fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the StrepFlag tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or StrepFlag-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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryo, Embryonic Stem Cell

DISEASE(S): Disease Free

SUBMITTER: Karsten Boldt  

LAB HEAD: Dr. Karsten Boldt

PROVIDER: PXD024680 | Pride | 2021-11-03

REPOSITORIES: Pride