the skeleton is in a dynamic state , being continually degraded and renewed in a tightly regulated remodeling process that involves a complex network of systemic hormones and local factors . among the local signaling factors implicated in this process opg , a secreted member of the tumor necrosis factor receptor superfamily , has been identified as an osteoblast - derived regulator of bone resorption and bone mass , and it is implicated in the pathogens of postmenopausal osteoporosis and other metabolic bone diseases . opg acts by neutralizing rankl , an essential cytokine required for osteoclast formation and activation . prior studies have showed that opg is not only an important regulatory factor of bone growth , bone modeling , and bone remodeling [ 2 , 3 ] , but also an intermediary factor of a wide variety of hormones , cytokine , and growth factors which are involved in regulating bone metabolism . in vitro , osteoclast differentiation which is induced by 1,25-(oh)2d3 , pth , pge2 , and il-4 is blocked by opg in a dose - dependent manner [ 5 , 6 ] ; in vivo , recombinant opg increases significantly bone mass and bone mineral density of lumbar spine and femoral of ovariectomized rat . in clinical trials , recombinant opg can be used for postmenopausal women for the treatment of osteoporosis . these data suggested that opg could act as a key factor in the regulation of bone mass and implied a utility for opg in the treatment of metabolic bone disease . in addition to the effect on bone tissue , opg was also reported to influence cardiovascular   and immune system . therefore , a suitable animal model with opg overexpression is needed for the further insight into the implication of opg in metabolic bone disease . transgenesis is a process where foreign genes are inserted into an animal 's dna , and it is popular in the biomedical science . scientists create , so - called transgenic animals to investigate disease treatments , produce natural material , and expand scientific knowledge . transgenic mice are the most widely used as experimental models to unravel gene phenotype and they are valuable tools for biomedical research . in our previous study , opg knockout mice showed increased bone remodeling , deceased bone density , and reduction in bone strength . in the present work , we firstly created opg transgenic mice containing human opg promoter and flag tag . furthermore , we obtained the quantitative data of bone microarchitecture in opg transgenic mice for the first time . c57bl/6j mice ( wt mice ) were purchased from the shanghai research center for biomodel organisms . all studies were performed with the approval of the experimental animal committee at the university of south china . all the animals were housed under specific pathogen - free conditions ( 22c , 12  h/12  h light / dark , 50%55% humidity ) with free access to food pellets and tap water in the experimental animal center of the university of south china . the liver rna extracted from c57bl/6j mice was used to perform two pcr amplification using pfu pcr enzyme to obtain mopg cdna sequence . then mopg cdna sequence and pci - neo - opg - lacz plasmid which contained human opg promoter were double digested by using not i and sal i. the digested fragments were connected by using t4 ligase , and then the connected fragments were transferred into competent cells from escherichia coli and tiled in lb plate . monoclonal bacteria were selected and the pci - hopgp - mopg plasmid was confirmed by enzyme digestion and sequence reaction . the vector was digested by using bglii / kpni to obtain linear dna fragment for further transgenic mice establishment . six  eight - week c57bl/6j  cba female mice were selected to perform ovarian stimulation and subsequently mated with healthy male mice overnight . the female mice which have vaginal pessary were selected as receptor for embryo transplantation , and then fertilized eggs were obtained from the receptors by surgery . linear dna was injected into fertilized eggs ( pronuclear stage ) by using an inverted microscope , micromanipulation equipment , and injection / holding devices . to generate opg - tg mice , 15~30 recombinant cells were injected into c57bl/6j blastocysts and then transferred into uterus of pseudopregnant mice . after young mice were born , progeny was screened for the presence of the opg by pcr and western blot . genomic dna was extracted and purified from the tail tissue of each mouse . to genotype the opg - tg mice , pcr was performed using the following primers : opg - jd-1  :  5-tca aag gca ggc gat act-3 and opg - jd-2  :  5-caa tgt ctt cct cct cac tgt-3 . the mice were sacrificed after 12 weeks , and proteins were extracted from the livers . the opg protein levels in the mice were detected by performing a western blot assay using rabbit anti - flag polyclonal antibodies [ 13 , 14 ] . the right tibia was fixed with 4% paraformaldehyde for 24  h and subsequently washed with 10% saccharose solution for 12  h. micro - ct scanning was performed using the ge explore locus sp system . the center of rotation and the ct value were artificially modified and the entire results obtained for the samples scanned with an isotropic resolution of 8.0  8.0  8.0  m voxels were reconstructed after completion of the scanning . for the cancellous bone analysis , bone tissue of 0.8  mm thickness at a distance of 0.16  mm from the distal end of the growth plate was selected from the image as the region of interest ( roi ) in order to perform a three - dimensional reconstruction of 8.0  8.0  8.0  m voxels ; image information was obtained based on the automatic domain value provided by the computer in order to accomplish binary conversion of the image . selected areas of cancellous bone within the roi were demonstrated three dimensionally . for the cortical bone analysis , bone tissue , 1.0  mm in length and 0.16  mm in thickness , from the middle diaphysis of the tibia was selected as the roi from the reconstructed image to perform three - dimensional reconstruction of 8.0  8.0  8.0  m voxels , and image information was obtained based on the automatic domain value yielded by the computer . the 2.0aba microview software of the micro - ct system was applied to perform quantitative analysis on the reconstructed images . the left femur was fixed onto the scanning table along the longitudinal axis and subsequently scanned by dual - energy x - ray absorptiometry ( dxa ) using a piximus densitometer ( ge lunar , madison , wi ) to determine the bmd and bmc . scanning parameters are as follows : the scan spacing was 1.0  1.0  mm , scanning speed was 60.0  mm / s , and the accuracy was 1.0% . data were analyzed by analysis of variance followed by the student - newman - keuls multiple comparisons test , and statistical significance was considered when p was less than 0.05 . the liver rna extracted from c57bl/6j mice was utilized to perform rt - pcr to obtain opg cdna sequence . opg cdna sequence and pci - neo - opg - lacz plasmid ( figure 1(a ) ) containing human opg promoter were double digested by using not i and sal i , and a 1.2  kb fragment from mopg cdna and a 10.4  kb fragment from pci - neo - opg - lacz were observed ( figures 2(a ) and 2(b ) ) . the recombinant pci - hopgp - mopg plasmid ( figure 1(b ) ) showed a 1.2  kb fragment and a 10.4  kb fragment with the digestion of not i and sal i. 766  bp , and 5354  bp and 5586  bp fragments were observed after the digestion ecor ( figure 2(c ) ) . the recombinant pci - hopgp - mopg plasmid was linealized by the digestion with bglii and kpni to obtain two dna fragments of 8291  bp and 3411  bp , and the 8291  bp linearized pci - hopgp - mopg fragment was microinjected into mouse fertilized egg cells ( figure 2(d ) ) . during the course of transgenic mice preparation , total 426 injection eggs were transplanted into 17 pseudopregnant female mice tuba , and 12 female mice of them were shown to be pregnant . total 69 mice were born and 7 positive transgenic mice were confirmed by pcr screening . furthermore , genomic dna extracted from positive mice and wt mice tails was used for double - blinded pcr to further confirm the seven positive transgenic mice ( figure 3(a ) ) . the transgenic founder female mice were fed together with wt male mice in the cage rearing at the ratio of 1  :  1 , and transgenic founder male mice were fed with wt female mice in cage at the ratio of 1  :  3 . to identify the genotype of the offspring , special primers across the hopg promoter and mopg were used for the pcr screening , and as shown in figure 3(b ) , no band was detected in wt mice and one 385  bp band was observed in opg - tg mice . to further confirm the genotype of the transgenic mice , the proteins were extracted from the mice livers and western blot was performed using rabbit anti - flag polyclonal antibodies . as shown in figure 3(c ) , the flag tag could not be detected in the wt mice , whereas a 60-kd opg protein containing flag tag could be detected in the opg - tg mice . it was found that opg - tg mice showed increases of 42.3% and 151.7% of overall femoral bmd and bmc , respectively , compared to those of wt mice ( table 1 ) . to observe the effect of opg on bone microarchitecture , micro - ct was used . as shown in figure 4 , compared with wt mice , cancellous bone trabecular number of opg - tg mice increased significantly ; distribution of bone trabecula in opg - tg mice became denser and more continuous and trabecular separation of opg - tg mice became smaller . micro - ct scanning also showed that the opg - tg mice had an increase of 40.6% , 11.7% , 60% , and 62.1% for vbmd , tbmd , tb.th , and tb.n , respectively , and a decrease of 47.4% for th.sp compared to those of wt mice ( table 2 ) . the cortical bone structural parameter also showed difference between opg - tg mice and wt mice . opg - tg mice showed obviously thicker cortical bone and narrower lumen ( figure 4 ) . it was also confirmed that the opg - tg mice mice showed an increase of 81.0% , 132.9% , 15.7% , and 118.2% for ct.ar , ct.th , ct.bmd , and ct.bmc respectively , and a decrease of 15.4% for in.pm compared to those of wt mice . no significant outer perimeter ( ot.pm ) difference was observed between opg - tg mice and wt mice ( table 3 ) . opg , a novel secreted member of the tnfr superfamily , acts as a soluble receptor antagonist of rankl by preventing it from binding to rank , and the interaction of rankl and rank has been shown to be required for osteoclast differentiation . any dysregulation of their respective expression leads to bone tumor - associated osteolysis , immune disease , and cardiovascular pathology . many researches have proved that opg can inhibit osteoclast maturation and protect bone from both normal osteoclast remodeling and ovariectomy - associated bone loss , indicating that opg might be a key determinant in regulating bone metabolism . firstly , rankl is a membrane - bound protein of the tumor necrosis factor ligand family that is expressed on the osteoblast cell surface and has been shown to play a major role in osteoclast differentiation along with macrophage colony stimulating factor . rankl binds to its receptor rank on hematopoietic cells and initiates a cascade of signaling events that leads to osteoclast differentiation . as a decoy receptor for rankl opg has been shown to be a potent inhibitor of bone resorption through interfering osteoclast survival , differentiation , and biological activity in vitro . secondly , opg can prevent taf6 activation with rank / rankl , thus inhibiting osteoclast activation and maturation , and this could ultimately result in the reduction of functional osteoclast . thirdly , opg can also prevent stroma cell from the interaction with osteoclast , and this inhibitory ability can be protected in the presence of caspase-3 inhibitor , indicating that opg can prevent bone loss through inducing osteoclast apoptosis . finally , opg can also affect the function of osteoclast directly by increasing the protease and trypsin inhibitor expression . in summary , the mechanism of opg in regulating bone metabolism is complicated and deserves more investigations . opg was also shown to influence cardiovascular and immune system except for the bone tissue . opg - deficient mice have shown early onset arterial calcification [ 18 , 19 ] , suggesting an important role of opg in the protection of blood vessels , whereas recent studies showed that opg might increase adhesion function of endothelial cell and serum opg level is associated with atherosclerosis severity . in the immunity system , opg can regulate dendritic cell differentiation and maturation , affecting lymphocyte development and function . due to the diverse function of opg , a suitable animal model is needed for the comprehensive understanding of opg function . recently , the advances in transgenic technologies have made it possible for the developments of different transgenic animal models . compared with other animal model , mouse shows higher genome homology with human , so it is widely used as transgenic animal model for biomedical research . hence , the mouse is unique in offering the possibility to understand genotype - phenotype relationships that are relevant for unraveling the biologic role of the genes in human . opg - deficient mice exhibited bone loss with an increase in both bone resorption and formation , which just like high bone turnover postmenopausal osteoporosis . in this work , we ligated full - length mouse opg cdna sequence with pci - neo - lacz plasmid which contains human opg promoter to obtain pci - hopgp - mopg vector . because variety of flag - tagged protein can retain their biochemical activity , flag tag is widely utilized as screening marker in the development of transgenic cells and animals models so flag tag was added to pci - hopgp - mopg vector to improve the screen efficiency of opg - tg mice . because human opg promoter has a transcriptional activity without tissue specificity , it could be anticipated that the established opg transgenic mice could have a widely exogenous expression of opg in different tissues . in order to avoid possible noise from endogenous opg in the screening process , specific primers that span from the human opg promoter to mopg were designed to improve the correct rate in the opg - tg mice screening ; moreover , flag protein expression in the opg - tg mice was also detected to further devoid those transgenic mice without opg expression due to different genomic context . since different founder mice had different insertion sites , we got at least two transgenic mice lines to observe their phenotype , and the same phenotypes from different transgenic mice lines were considered as phenotypes of opg gene .   micro - ct is an evolving technique that has the ability to measure three - dimensional ( 3d ) bone microstructure in arbitrary orientations in a highly automated , objective , non - destructive manner , allowing great number of samples for unbiased comparisons between controls and the disordered or treated . compared to two - dimensional image ( 2d ) data of dxa , micro - ct can not only directly observe the 3d images of cortical bone and trabecular bone microstructure , but also make a quantitative analysis of the 3d structure such as trabecular volume , trabecular thickness , number , separation , structure model index , degree of anisotropy , and connectivity , in a model - independent manner . quantitative analysis of 3d bone microstructure characteristics may improve our ability to understand the pathophysiology of osteoporosis , to test the efficacy of pharmaceutical intervention , and to estimate bone biomechanical properties . so it is very meaningful to measure 3d bone microstructure of opg transgenic mice to observe the direct effect of opg on bone mass . previous research has reported the quantitative analysis of 3d bone microstructure in the opg transgenic rats , and in the present study , we for the first time quantified 3d bone microstructure in opg transgenic mice . micro - ct data showed that compared with wild - type mice , the opg - tg mice showed increased tibial cancellous bone microstructural parameters such as vbmd , tbmd , tb.th , and tb.n than those in wild - type mice . the cortical bone microarchitecture parameters of tibia such as ct.ar , ct.th , ct.bmd , and ct.bmc in opg - tg mice were significantly higher than those in wt mice . the 3d structure diagram of tibia showed that cortical bone appeared thick and luminal appeared narrow in opg - tg mice . the 2d image data of dxa also showed that bmd and bmc were significantly improved in opg - tg mice . in summary , we for the first time developed opg transgenic mice model containing human opg promoter and flag tag , and a quantitative analysis of the 3d bone microarchitecture in opg transgenic mice was also carried out . our results show that the bone mass of cancellous and cortical bone in opg transgenic mice is significantly increased . the established opg transgenic mouse model would offer a suitable animal model for further insight into the regulating mechanism of bone metabolism .
osteoprotegerin ( opg ) plays a determinant role in regulating bone metabolism , but the effect of opg on bone microarchitecture needs to be further elucidated . we attempted to construct pci - hopgp - mopg vector containing human opg promoter and flag tag and to microinject vector into fertilized zygotes from c57bl/6j    cba mice to prepare transgenic mice . 
 the opg transgenic positive mice were identified by pcr and western blotting . 
 twelve - week - old opg transgenic mice ( opg - tg mice ) and wild - type mice ( wt mice ) were utilized in the study of bone microarchitecture . 
 microcomputed tomography ( micro - ct ) data showed that compared with wt mice , the tibia of opg - tg mice showed an increased volumetric bmd ( vbmd ) , tissue bmd ( tbmd ) , trabecular thickness ( tb.th ) , and trabecular number ( tb.n ) , and a decreased trabecular separation ( th.sp ) ( p < 0.05 ) . 
 the cortical bone microarchitecture parameters , such as cortical area ( ct.ar ) , cortical thickness ( ct.th ) , cortical bmd ( ct.bmd ) , cortical bmc ( ct.bmc ) , bmd , and bmc of femur , were increased , and the inner perimeter ( in.pm ) was decreased , in opg - tg mice , compared to those in wt mice ( p < 0.05 ) . 
 the established opg transgenic mouse model could be valuable for further studying the biological significance and gene regulation of opg in vivo .