IADR Abstract Archives
Characterization of the PLAP-1-GFP/CreER knock-in mouse
Abstract: Objectives:
Periodontal ligament (PDL) is the fibrous connective tissue between the alveolar bone and cementum. The function of PDL cells was studied extensively in vitro, however, the molecular regulations for homeostasis of PDL in vivo remain elucidative. The inducible Cre-lox system is a valuable tool to study the regulation of the tissue homeostasis in a spatial and time-restricted fashion. We have previously found that PLAP-1/Aspn is highly and specifically expressed in PDL. In this study, we aimed to generate and characterize a mouse line that expresses both green fluorescent protein (GFP) and tamoxifen-inducible Cre recombinase from endogenous PLAP-1 locus.
Methods:
A targeting construct was created that inserts the CreERT2-2A-EGFP-WPRE cassette into the endogenous start codon of the PLAP-1 gene. The construct was electroporated into C57BL/6 embryonic stem (ES) cells, and correctly targeted ES cells were injected into C57BL/6 blastocysts, which were implanted into pseudo-pregnant females. The resulting chimeric mice were bred with C57BL/6, and offspring were analyzed. EGFP expression in periodontal tissue was compared with PLAP-1 endogenous mRNA expression by RNAscope in situ hybridization. Further, the knock-in mice were crossed with Rosa26-tdTomato Cre reporter mice to label PLAP-1 lineage cells. After the development of the periodontium was completed, the mice are treated with Tamoxifen.
Results:
Newly established PLAP-1-GFP/CreER knock-in mouse expressed GFP in most spindle-shaped fibroblastic cells but not in osteoblasts, cementoblasts, and blood vessels in PDL. These signals were matched to in situ expressions of PLAP-1 mRNA expression. Upon crossing with Cre reporter mice and 3-day after tamoxifen treatment, tdTomato expression was matched to GFP expression.
Conclusions:
The fluorescent reporter and lineage tracing analysis showed faithful expression of GFP/CreER under the PLAP-1 promoter in the knock-in mouse. This method can be a valuable tool to i) trace the fate of PDL cells in vivo by crossing with Cre reporter, ii) study gene functions in PDL by crossing with mice with the floxed allele of the targeted gene, and iii) isolate pure PDL cells according to their GFP expression.
Periodontal ligament (PDL) is the fibrous connective tissue between the alveolar bone and cementum. The function of PDL cells was studied extensively in vitro, however, the molecular regulations for homeostasis of PDL in vivo remain elucidative. The inducible Cre-lox system is a valuable tool to study the regulation of the tissue homeostasis in a spatial and time-restricted fashion. We have previously found that PLAP-1/Aspn is highly and specifically expressed in PDL. In this study, we aimed to generate and characterize a mouse line that expresses both green fluorescent protein (GFP) and tamoxifen-inducible Cre recombinase from endogenous PLAP-1 locus.
Methods:
A targeting construct was created that inserts the CreERT2-2A-EGFP-WPRE cassette into the endogenous start codon of the PLAP-1 gene. The construct was electroporated into C57BL/6 embryonic stem (ES) cells, and correctly targeted ES cells were injected into C57BL/6 blastocysts, which were implanted into pseudo-pregnant females. The resulting chimeric mice were bred with C57BL/6, and offspring were analyzed. EGFP expression in periodontal tissue was compared with PLAP-1 endogenous mRNA expression by RNAscope in situ hybridization. Further, the knock-in mice were crossed with Rosa26-tdTomato Cre reporter mice to label PLAP-1 lineage cells. After the development of the periodontium was completed, the mice are treated with Tamoxifen.
Results:
Newly established PLAP-1-GFP/CreER knock-in mouse expressed GFP in most spindle-shaped fibroblastic cells but not in osteoblasts, cementoblasts, and blood vessels in PDL. These signals were matched to in situ expressions of PLAP-1 mRNA expression. Upon crossing with Cre reporter mice and 3-day after tamoxifen treatment, tdTomato expression was matched to GFP expression.
Conclusions:
The fluorescent reporter and lineage tracing analysis showed faithful expression of GFP/CreER under the PLAP-1 promoter in the knock-in mouse. This method can be a valuable tool to i) trace the fate of PDL cells in vivo by crossing with Cre reporter, ii) study gene functions in PDL by crossing with mice with the floxed allele of the targeted gene, and iii) isolate pure PDL cells according to their GFP expression.