title: Effects of osteoprotegerin from transfection of pcDNA3.1(+)/chOPG on bioactivity of chicken osteoclasts.
abstract: BACKGROUND
  Osteoprotegerin (OPG) has been reported to prevent bone resorption by inhibiting the formation, function, and survival of osteoclasts in a variety of animal models. However, the effects of OPG on bone metabolism in avian species have not been described. The objective of this study was to investigate the effects of chicken OPG (chOPG) expressed in chicken embryo fibroblasts (CEFs) on chicken osteoclast function in vitro.
  
  
  METHODS
  The chOPG sequence containing the open reading frame (ORF) was amplified from chicken embryo frontal bone and inserted into the pcDNA3.1 (+) vector. PcDNA3.1 (+)/chOPG was transiently transfected into CEFs by lipofectamine 2000. Transcription of OPG mRNA and expression of chOPG recombinant protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence. The level of chOPG recombinant protein was detected by enzyme-linked immunosorbent assay. The suspension of osteoclasts was separated from chicken embryos and divided into three groups (control group, pcDNA3.1 (+) group and pcDNA3.1 (+)/chOPG group). The percentage of osteoclast apoptosis was detected by flow cytometry. The tartrate-resistant acid phosphatase (TRAP) secreted by osteoclasts was measured by the diazol method. The resorbing activity of osteoclasts was evaluated by the area of lacunae on bone flaps and the concentration of calcium in the supernatant liquid of osteoclasts.
  
  
  RESULTS
  48 h after transfection, the exogenous OPG gene transcription was detected by RT-PCR. After 72 h, the CEFs transfected from pcDNA3.1 (+)/chOPG displayed green fluorescence and the concentration of chOPG protein was 15.78 ± 0.22 ng/mL. After chicken osteoclasts were cultured for 5 d in a medium containing supernatant from transfected CEFs, the percentage of osteoclast apoptosis was increased significantly, the concentration of TRAP, the area of lacunae on bone flaps and calcium concentration were decreased significantly in the pcDNA3.1(+)/OPG group compared to the control group and the pcDNA3.1 (+) group.
  
  
  CONCLUSIONS
  Constructed pcDNA3.1 (+)/chOPG transfected into CEFs expressed bioactive OPG protein that was able to inhibit osteoclast function.
authors: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China.
authors: Lele
authors: L
authors: Hou
authors: Jiafa
authors: J
authors: Hou
authors: Jing
authors: J
authors: Yao
authors: Zhenlei
authors: Z
authors: Zhou
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D010010:Osteoclasts / Q000166:cytology / Q000502:physiology*; D053244:Osteoprotegerin / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D053244:Osteoprotegerin; D011994:Recombinant Proteins