During bone development and repair, osteoblasts get recruited to bone deposition sites. To identify the origin of recruited osteoblasts, cell lineage tracing using Cre/loxP recombination is commonly utilized. However, a confounding factor is the use of transgenic Cre drivers that do not accurately recapitulate endogenous gene expression or knock-in Cre drivers that alter endogenous protein activity or levels. Here, we describe a novel CRISPR/Cas9 homology-directed repair knock-in approach that allows efficient generation of Cre drivers controlled by the endogenous gene promoter. Also, a self-cleaving peptide preserves reading frame of the endogenous protein. Using this approach, we generated col10a1p2a-CreERT2 knock-in medaka and showed that tamoxifen-inducible CreERT2 efficiently recombined loxP sites in col10a1 cells. Similar knock-in efficiencies were obtained when two unrelated loci (osr1, col2a2) were targeted. Using live imaging, we traced the fate of col10a1 osteoblast progenitors during bone lesion repair in the medaka vertebral column. We show that col10a1 cells at neural arches represent a mobilizable cellular source for bone repair. Together, our study describes a novel strategy for precise cell lineage tracing via efficient and non-disruptive knock-in of Cre.
A novel non-disruptive and efficient knock-in allows fate tracing of resident osteoblast progenitors during repair of vertebral lesions in medaka
- Award Group:
- Funder(s): Ministry of Education - Singapore
- Award Id(s): MOE2016-T2-2-086
- Funder(s):
- Award Group:
- Funder(s): National Research Foundation Singapore
- Award Id(s): NRF2017-NRF-ISF002-2671
- Funder(s):
Currently Viewing Accepted Manuscript - Newer Version Available
Tan Wen Hui, Christoph Winkler; A novel non-disruptive and efficient knock-in allows fate tracing of resident osteoblast progenitors during repair of vertebral lesions in medaka. Development 2022; dev.200238. doi: https://doi.org/10.1242/dev.200238
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