Abstract
Nkx6-1 is a member of the Nkx family of homeodomain transcription factors (TFs) that regulates motor neuron development, neuron specification and pancreatic endocrine and β-cell differentiation. To facilitate the isolation and tracking of Nkx6-1-expressing cells, we have generated a novel Nkx6-1 Venus fusion (Nkx6-1-VF) reporter allele. The Nkx6-1-VF knock-in reporter is regulated by endogenous cis-regulatory elements of Nkx6-1 and the fluorescent protein fusion does not interfere with the TF function, as homozygous mice are viable and fertile. The nuclear localization of Nkx6-1-VF protein reflects the endogenous Nkx6-1 protein distribution. During embryonic pancreas development, the reporter protein marks the pancreatic ductal progenitors and the endocrine lineage, but is absent in the exocrine compartment. As expected, the levels of Nkx6-1-VF reporter are upregulated upon β-cell differentiation during the major wave of endocrinogenesis. In the adult islets of Langerhans, the reporter protein is exclusively found in insulin-secreting β-cells. Importantly, the Venus reporter activities allow successful tracking of β-cells in live-cell imaging and their specific isolation by flow sorting. In summary, the generation of the Nkx6-1-VF reporter line reflects the expression pattern and dynamics of the endogenous protein and thus provides a unique tool to study the spatio-temporal expression pattern of this TF during organ development and enables isolation and tracking of Nkx6-1-expressing cells such as pancreatic β-cells, but also neurons and motor neurons in health and disease.
Highlights
Introduction published maps and institutional affilThe endoderm-derived pancreas comprises exocrine and endocrine compartments that contribute to nutrient digestion and regulate blood glucose homeostasis, respectively
The Nkx6-1-VF mouse line was generated by CRISPR/Cas9-mediated double strand breaks followed by homologous recombination
A loxP-flanked phosphoglycerate kinase (PGK) promoter-driven neomycin (Neo) resistance gene in the opposite orientation was inserted after the reporter gene
Summary
The Nkx6-1-VF mouse line was generated by CRISPR/Cas9-mediated double strand breaks followed by homologous recombination. Using a targeting vector as template DNA for directed repair resulted in the generation of an Nkx6-1-VF reporter gene under control of the endogenous Nkx cis-regulatory elements (Figure 1a). We designed a targeting vector by standard cloning techniques by removing the translational stop codon of the Nkx gene in exon 3 and generating an in-frame fusion transcript with the. The loxP-flanked Neo selection cassette was deleted in the germline by Cre recombination-mediated excision (Figure 1d; [27]). No embryonic phenotype was observed and offspring homozygous for the Nkx6-1-VF allele were viable and appeared indistinguishable from their wild-type littermates. We showed that the reporter is translated as a fusion protein and can be detected with Flag and Venus antibodies
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