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. 2021 Sep 28;14(1):383.
doi: 10.1186/s13104-021-05785-4.

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

Karin Schuster-Gossler  1 , Karsten Boldt  2 , Dorothee Bornhorst  1   3   4   5 , Patricia Delany-Heiken  1 , Marius Ueffing  2 , Achim Gossler  6
Affiliations

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

Karin Schuster-Gossler et al. BMC Res Notes. .

Abstract

Objective: 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.

Results: 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: AcGFP; DLL1 C-terminal tagging; DLL1 hypomorph; Mouse DLL1; StrepFLAG.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of DLL1AcGFPHA and DLL1SF proteins in CHO cells. A Scheme of constructs used to express DLL1AcGFPHA or DLL1SF in CHO cells under control of the CMV2 promoter. Full length construct (top), 3’ region before (middle), and after (bottom) Cre-mediated recombination. B Expression of DLL1 variants in CHO cells carrying DLL1AcGFPHA or DLL1SF. DLL1 variants were immunoprecipitated with anti HA (a) anti-GFP (b) or anti-Flag (c) antibodies and detected in Western blots using anti-DLL1 antibody 1F9. + control: Lysate of CHO cells stably overexpressing DLL1. Arrows point to tagged DLL1 proteins. Photographs of the Western blot membranes are shown in Additional file 7: Figure S3. C AcGFP fluorescence in DLL1AcGFPHA expressing CHO cells (row a) in comparison to wild-type CHO cells (row b); Column I: fluorescence, II: overlay, III: bright field. D Surface presentation of DLL1AcGFPHA (dots) and DLL1SF (triangles) in CHO detected in cell surface biotinylation assays
Fig. 2
Fig. 2
Analysis of Dll1AcGFPstopSF and Dll1SF mice. A Scheme of the modified 3’ region before (top) and after (bottom) Cre-mediated recombination. B Phenotype of wt (a), homozygous adult Dll1AcGFPHAstopSF (b) and Dll1SF (c) mice. C Skeletal preparations of wt (a), homozygous Dll1GFPHAstopSF (b) and Dll1SF d15.5 fetuses. D Detection of DLL1 variants in homozygous Dll1AcGFPHAstopSF, Dll1SF and wild type d10.5 embryos. DLL1 variants were immunoprecipitated with anti-HA (a), anti-GFP (b) or anti-Flag (c) antibodies and detected in Western blots using anti-DLL1antibody 1F9. + control: Lysate of DLL1 overexpressing ES cells. Arrows point to tagged DLL1 proteins. Photographs of the Western blot membranes are shown in Additional file 8: Figure S4. E GFP fluorescence in homozygous d9,5 Dll1AcGFPHAstopSF (a–a’’) and wild type (b–b’’) embryos; (a, b) bright field, (a’, b’) fluorescence, (a’’, b’’) overlay. Arrows in (a) point to irregular somites
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