A non-disruptive and efficient knock-in approach allows fate tracing of resident osteoblast progenitors during repair of vertebral lesions in medaka.
Wen Hui TanChristoph WinklerPublished in: Development (Cambridge, England) (2022)
During bone development and repair, osteoblasts are recruited to bone deposition sites. To identify the origin of recruited osteoblasts, cell lineage tracing using Cre/loxP recombination is commonly used. However, a confounding factor is the use of transgenic Cre drivers that do not accurately recapitulate endogenous gene expression or the use of knock-in Cre drivers that alter endogenous protein activity or levels. Here, we describe a CRISPR/Cas9 homology-directed repair knock-in approach that allows efficient generation of Cre drivers controlled by the endogenous gene promoter. In addition, a self-cleaving peptide preserves the reading frame of the endogenous protein. Using this approach, we generated col10a1p2a-CreERT2 knock-in medaka and show that tamoxifen-inducible CreERT2 efficiently recombined loxP sites in col10a1 cells. Similar knock-in efficiencies were obtained when two unrelated loci (osr1 and col2a1a) 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 previously unreported strategy for precise cell lineage tracing via efficient and non-disruptive knock-in of Cre.
Keyphrases
- bone mineral density
- gene expression
- bone regeneration
- single cell
- induced apoptosis
- crispr cas
- postmenopausal women
- soft tissue
- dna methylation
- cell cycle arrest
- bone loss
- body composition
- genome editing
- cancer therapy
- oxidative stress
- working memory
- drug delivery
- cell death
- protein protein
- endoplasmic reticulum stress
- copy number
- breast cancer cells
- liquid chromatography
- quality improvement
- pi k akt
- photodynamic therapy
- emergency medicine