Generation And Phenotypic Analysis Of Ppp2ca Conditional Knockout Mice In Osteoblasts

Background and Objective:Protein phosphatase 2A?PP2A?,as one of the main serine/threonine phosphatases in mammals,is involved in regulating most of the phosphorylation in eukaryotic cells.PP2A takes part in multiple pathways and influences many physiological process including cell cycle progression,apoptosis,DNA replication and transcription.Recent research shows that PP2A,especially the catalytic C subunit?Ppp2ca?,regulates bone formation and osteoblast differentiation via bone formation related Osterix gene in cellular level.The research aims at constructing the tool mice that can specifically express CRE recombinase in osteoblasts,and obtaining the mice with Ppp2ca conditional knockout in osteoblasts to explore the effect of Ppp2ca in bone formation in mice.Method:Constructed the vector pNCHS4-Col1-Cre-NLs-BPA,and obtained the transgenic tool mice which could express CRE enzyme in osteoblasts?Col1-Cre?through pronuclear injection and PCR identification.X-gal staining was used to detect the activity of CRE enzyme in osteoblasts in the offspring's which obtained from the mating of Col1-Cre mice to ROSA26 reporter mice.The Col1-Cre mice were crossed with Ppp2ca conditional null allele mice(Ppp2ca^fl/fl)for two generations to get Ppp2ca osteoblasts conditional knockout mice.The effect of Ppp2ca in bone formation in mice was explored through Western,HE staining,X-ray and Micro-CT bone mineral density?BMD?analysis.Results:The Col1-Cre^T/W/W mice were obtained through pronuclear injection and identified by PCR.The mice with both Col1-Cre and LacZ gene(Col1-Cre^T/W;ROSA26^T/W)were acquired by the crossing of Col1-Cre^T/W/W mice and ROSA26^T/W/W mice.The CRE enzyme expressed successfully in mice osteoblasts proving by frozen section and X-gal staining of Col1-Cre^T/W;ROSA26^T/W mice.In this research,the mice with Ppp2ca conditional knockout in osteoblasts were obtained,and Ppp2ca was proved to be knockout in osteoblasts by PCR and western blotting.We contrasted the knockout mouse with the litter control.Then we find 20%of the knockout mice were obviously smaller than its litter controls with no significant difference in the size and length of femur.The bone cortex showed fragmentation via HE staining of mice femur after 3 days decalcification,and more cracks in bone cortex and thicker ossification area from epiphysis to Periosteum after 7 days decalcification.It was supposed that the knockout of Ppp2ca in osteoblasts brought about a higher activity of endochondral ossification.X-ray was used to find out obviously different group to do Micro-CT scan.The knockout mice showed higher BMD of cortical bone and lower BMD of trabecular bone through BMD analysis,while the obviously smaller knockout mice showed lower BMD of both cortical and trabecular bone.Conclusion:This research successfully structured Col1-Cre transgenic tool mice and obtained Ppp2ca osteoblasts conditional knockout mice.Throgh the phenotype analysis of Col1-Cre^T/W;Ppp2ca^fl/fl mice,it was proved that Ppp2ca deletion in osteoblasts led to higher activity of endochondral ossification and BMD of mice femur.