Our previous studies have demonstrated that Fam20 C promotes differentiation and mineralization of odontoblasts,ameloblasts,osteoblasts and osteocytes during tooth and bone development.Ablation of the Fam20 C gene inh...Our previous studies have demonstrated that Fam20 C promotes differentiation and mineralization of odontoblasts,ameloblasts,osteoblasts and osteocytes during tooth and bone development.Ablation of the Fam20 C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice.However,control and regulation of the expression of Fam20 C are still unknown.In this study,we generated a transgenic reporter model which expresses green fluorescence protein（GFP） driven by the Fam20 C promoter.Recombineering was used to insert a 16 kb fragment of the mouse Fam20 C gene（containing the 15 kb promoter and 1.1 kb of exon 1） intoa pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence.GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice.Fluorescence was evident in the bone and teeth as early as E17.5.The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth.The expression of GFP was significantly reduced in teeth,alveolar bone and muscle by 8 weeks of age.We also observed colocalization of the GFP signal with the Fam20 C antibody in postnatal 1- and 7-day-old animals.Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20 C gene expression and the biological function of this gene during odontogenesis and osteogenesis.展开更多
为了研究褪黑素受体在人脑中的表达状况以及褪黑素在老年性痴呆中的作用 ,本研究克隆了 Mel1 a和 RORβ等两种褪黑素受体的 c DNA并将其转入 p Bluescript ( KS+)质粒载体中。重组质粒通过限制性内切酶鉴定分析并测序。结果表明 ,克隆...为了研究褪黑素受体在人脑中的表达状况以及褪黑素在老年性痴呆中的作用 ,本研究克隆了 Mel1 a和 RORβ等两种褪黑素受体的 c DNA并将其转入 p Bluescript ( KS+)质粒载体中。重组质粒通过限制性内切酶鉴定分析并测序。结果表明 ,克隆的序列与 Gen Bank中的序列一致。利用线性化的质粒模板反转出 c RNA探针 。展开更多
Based on the protein comparison between zebrafish Gli2 and fruit fly C_i and the analysis of hydrophilicity/hydrophobicity of Gli2 protein by computer programmer, the recombination N-terminal partial protein of Gli2 p...Based on the protein comparison between zebrafish Gli2 and fruit fly C_i and the analysis of hydrophilicity/hydrophobicity of Gli2 protein by computer programmer, the recombination N-terminal partial protein of Gli2 protein was designed.After the DNA fragment encoding the N-terminal part was cloned out by PCR, it was ligated with an expression vector containing His-tag and transformed into BL-21, a bacterial expression strain, to express this protein.After the expression condition was optimized, the protein can be expressed at high level after having been induced for 3 hours when the inducer—IPTG was at 0.6 mmol/L.The protein is soluble in solution, a bacterial protein extraction solution.After the protein solution was diluted in B-per solution, the recombination protein can bind to the Ni-NTA column and be purified highly.展开更多
基金supported by UCONN Health Center Startup Fund(Jian-Jun Hao)the American Association of Orthodontists Foundation(AAOF) (Jian-Jun Hao)
文摘Our previous studies have demonstrated that Fam20 C promotes differentiation and mineralization of odontoblasts,ameloblasts,osteoblasts and osteocytes during tooth and bone development.Ablation of the Fam20 C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice.However,control and regulation of the expression of Fam20 C are still unknown.In this study,we generated a transgenic reporter model which expresses green fluorescence protein（GFP） driven by the Fam20 C promoter.Recombineering was used to insert a 16 kb fragment of the mouse Fam20 C gene（containing the 15 kb promoter and 1.1 kb of exon 1） intoa pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence.GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice.Fluorescence was evident in the bone and teeth as early as E17.5.The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth.The expression of GFP was significantly reduced in teeth,alveolar bone and muscle by 8 weeks of age.We also observed colocalization of the GFP signal with the Fam20 C antibody in postnatal 1- and 7-day-old animals.Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20 C gene expression and the biological function of this gene during odontogenesis and osteogenesis.
文摘为了研究褪黑素受体在人脑中的表达状况以及褪黑素在老年性痴呆中的作用 ,本研究克隆了 Mel1 a和 RORβ等两种褪黑素受体的 c DNA并将其转入 p Bluescript ( KS+)质粒载体中。重组质粒通过限制性内切酶鉴定分析并测序。结果表明 ,克隆的序列与 Gen Bank中的序列一致。利用线性化的质粒模板反转出 c RNA探针 。
文摘Based on the protein comparison between zebrafish Gli2 and fruit fly C_i and the analysis of hydrophilicity/hydrophobicity of Gli2 protein by computer programmer, the recombination N-terminal partial protein of Gli2 protein was designed.After the DNA fragment encoding the N-terminal part was cloned out by PCR, it was ligated with an expression vector containing His-tag and transformed into BL-21, a bacterial expression strain, to express this protein.After the expression condition was optimized, the protein can be expressed at high level after having been induced for 3 hours when the inducer—IPTG was at 0.6 mmol/L.The protein is soluble in solution, a bacterial protein extraction solution.After the protein solution was diluted in B-per solution, the recombination protein can bind to the Ni-NTA column and be purified highly.
