Characterization of a novel murine Sost ER~(T2) Cre model targeting osteocytes
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摘要
Transgenic mice are widely used to delete or overexpress genes in a cell specific manner to advance knowledge of bone biology,function and disease.While numerous Cre models exist to target gene recombination in osteoblasts and osteoclasts,few target osteocytes specifically,particularly mature osteocytes.Our goal was to create a spatial and temporal conditional Cre model using tamoxifen to induce Cre activity in mature osteocytes using a Bac construct containing the 5' and 3' regions of the Sost gene(Sost ER~(T2) Cre).Four founder lines were crossed with the Ai9 Cre reporter mice.One founder line showed high and specific activity in mature osteocytes.Bones and organs were imaged and fluorescent signal quantitated.While no activity was observed in 2 day old pups,by 2 months of age some osteocytes were positive as osteocyte Cre activity became spontaneous or ‘leaky' with age.The percentage of positive osteocytes increased following tamoxifen injection,especially in males,with 43% to 95% positive cells compared to 19% to 32% in females.No signal was observed in any bone surface cell,bone marrow,nor in muscle with or without tamoxifen injection.No spontaneous signal was observed in any other organ.However,with tamoxifen injection,a few positive cells were observed in kidney,eye,lung,heart and brain.All other organs,28 in total,were negative with tamoxifen injection.However,with age,a muscle phenotype was apparent in the Sost-ERT2 Cre mice.Therefore,although this mouse model may be useful for targeting gene deletion or expression to mature osteocytes,the muscle phenotype may restrict the use of this model to specific applications and should be considered when interpreting data.
Transgenic mice are widely used to delete or overexpress genes in a cell specific manner to advance knowledge of bone biology,function and disease.While numerous Cre models exist to target gene recombination in osteoblasts and osteoclasts,few target osteocytes specifically,particularly mature osteocytes.Our goal was to create a spatial and temporal conditional Cre model using tamoxifen to induce Cre activity in mature osteocytes using a Bac construct containing the 5' and 3' regions of the Sost gene(Sost ER~(T2) Cre).Four founder lines were crossed with the Ai9 Cre reporter mice.One founder line showed high and specific activity in mature osteocytes.Bones and organs were imaged and fluorescent signal quantitated.While no activity was observed in 2 day old pups,by 2 months of age some osteocytes were positive as osteocyte Cre activity became spontaneous or ‘leaky' with age.The percentage of positive osteocytes increased following tamoxifen injection,especially in males,with 43% to 95% positive cells compared to 19% to 32% in females.No signal was observed in any bone surface cell,bone marrow,nor in muscle with or without tamoxifen injection.No spontaneous signal was observed in any other organ.However,with tamoxifen injection,a few positive cells were observed in kidney,eye,lung,heart and brain.All other organs,28 in total,were negative with tamoxifen injection.However,with age,a muscle phenotype was apparent in the Sost-ERT2 Cre mice.Therefore,although this mouse model may be useful for targeting gene deletion or expression to mature osteocytes,the muscle phenotype may restrict the use of this model to specific applications and should be considered when interpreting data.
Transgenic mice are widely used to delete or overexpress genes in a cell specific manner to advance knowledge of bone biology,function and disease.While numerous Cre models exist to target gene recombination in osteoblasts and osteoclasts,few target osteocytes specifically,particularly mature osteocytes.Our goal was to create a spatial and temporal conditional Cre model using tamoxifen to induce Cre activity in mature osteocytes using a Bac construct containing the 5' and 3' regions of the Sost gene(Sost ER~(T2) Cre).Four founder lines were crossed with the Ai9 Cre reporter mice.One founder line showed high and specific activity in mature osteocytes.Bones and organs were imaged and fluorescent signal quantitated.While no activity was observed in 2 day old pups,by 2 months of age some osteocytes were positive as osteocyte Cre activity became spontaneous or ‘leaky' with age.The percentage of positive osteocytes increased following tamoxifen injection,especially in males,with 43% to 95% positive cells compared to 19% to 32% in females.No signal was observed in any bone surface cell,bone marrow,nor in muscle with or without tamoxifen injection.No spontaneous signal was observed in any other organ.However,with tamoxifen injection,a few positive cells were observed in kidney,eye,lung,heart and brain.All other organs,28 in total,were negative with tamoxifen injection.However,with age,a muscle phenotype was apparent in the Sost-ERT2 Cre mice.Therefore,although this mouse model may be useful for targeting gene deletion or expression to mature osteocytes,the muscle phenotype may restrict the use of this model to specific applications and should be considered when interpreting data.
引文
1.Dallas,S.L.,Prideaux,M.&Bonewald,L.F.The osteocyte:an endocrine cell…and more.Endocr.Rev.34,658-690(2013).
2.Javaheri,B.et al.Deletion of a single beta-catenin allele in osteocytes abolishes the bone anabolic response to loading.J.Bone Miner.Res.29,705-715(2014).
3.Tatsumi,S.et al.Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction.Cell.Metab.5,464-475(2007).
4.Das,S.&Sakthiswary,R.Bone metabolism and histomorphometric changes in murine models treated with sclerostin antibody:a systematic review.Curr.Drug.Targets.14,1667-1674(2013).
5.Padhi,D.,Jang,G.,Stouch,B.,Fang,L.&Posvar,E.Single-dose,placebo-controlled,randomized study of AMG 785,a sclerostin monoclonal antibody.J.Bone Miner.Res.26,19-26(2011).
6.Delmas,P.D.Clinical potential of RANKL inhibition for the management of postmenopausal osteoporosis and other metabolic bone diseases.J.Clin.Densitom.11,325-338(2008).
7.Shimada,T.&Fukumoto,S.FGF23 as a novel therapeutic target.Adv.Exp.Med.Biol.728,158-170(2012).
8.Elefteriou,F.&Yang,X.Genetic mouse models for bone studies--strengths and limitations.Bone 49,1242-1254(2011).
9.Kalajzic,I.et al.In vitro and in vivo approaches to study osteocyte biology.Bone54,296-306(2013).
10.Dallas,S.L.,Xie,Y.,Shiflett,L.A.&Ueki,Y.Mouse Cre Models for the Study of Bone Diseases.Curr.Osteoporos.Rep.16,466-477(2018).
11.Abou-Khalil,R.&Colnot,C.Cellular and molecular bases of skeletal regeneration:what can we learn from genetic mouse models?Bone 64,211-221(2014).
12.Yang,W.et al.Dentin matrix protein 1 gene cis-regulation:use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo.J.Biol.Chem.280,20680-20690(2005).
13.Lu,Y.et al.DMP1-targeted Cre expression in odontoblasts and osteocytes.J.Dent.Res.86,320-325(2007).
14.Gorski,J.P.et al.Deletion of Mbtps1(Pcsk8,S1p,Ski-1)Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age.J.Biol.Chem.291,4308-4322(2016).
15.Lim,J.,Burclaff,J.,He,G.,Mills,J.C.&Long,F.Unintended targeting of Dmp1-Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice.Bone Res.5,16049(2017).
16.Bivi,N.et al.Cell autonomous requirement of connexin 43 for osteocyte survival:consequences for endocortical resorption and periosteal bone formation.J.Bone Miner.Res.27,374-389(2012).
17.Powell,W.F.Jr et al.Targeted ablation of the PTH/PTHrP receptor in osteocytes impairs bone structure and homeostatic calcemic responses.J.Endocrinol.209,21-32(2011).
18.Brunkow,M.E.et al.Bone dysplasia sclerosteosis results from loss of the SOSTgene product,a novel cystine knot-containing protein.Am.J.Hum.Genet.68,577-589(2001).
19.Balemans,W.et al.Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein(SOST).Hum.Mol.Genet.10,537-543(2001).
20.Balemans,W.&Van Hul,W.Extracellular regulation of BMP signaling in vertebrates:a cocktail of modulators.Dev.Biol.250,231-250(2002).
21.van Bezooijen,R.L.et al.Sclerostin is an osteocyte-expressed negative regulator of bone formation,but not a classical BMP antagonist.J.Exp.Med.199,805-814(2004).
22.van Bezooijen,R.L.et al.Sclerostin in mineralized matrices and van Buchem disease.J.Dent.Res.88,569-574(2009).
23.Winkler,D.G.et al.Osteocyte control of bone formation via sclerostin,a novel BMP antagonist.EMBO J.22,6267-6276(2003).
24.Poole,K.E.et al.Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation.FASEB J.19,1842-1844(2005).
25.Xiong,J.et al.Osteocytes,not Osteoblasts or Lining Cells,are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone.PLoS One 10,e0138189(2015).
26.Amrein,K.et al.Sclerostin and its association with physical activity,age,gender,body composition,and bone mineral content in healthy adults.J.Clin.Endocrinol.Metab.97,148-154(2012).
27.M?dder,U.I.et al.Relation of age,gender,and bone mass to circulating sclerostin levels in women and men.J.Bone Miner.Res.26,373-379(2011).
28.Moester,M.J.,Papapoulos,S.E.,L?wik,C.W.&van Bezooijen,R.L.Sclerostin:current knowledge and future perspectives.Calcif.Tissue Int.87,99-107(2010).
29.Zimmermann,C.et al.Histological characterization and biochemical analysis of paraspinal muscles in neuromuscularly healthy subjects.Muscle Nerve 52,45-54(2015).
30.Lynch,G.S.,Hinkle,R.T.,Chamberlain,J.S.,Brooks,S.V.&Faulkner,J.A.Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old.J.Physiol.535(Pt 2),591-600(2001).
31.Thornton,A.M.et al.Store-operated Ca(2+)entry(SOCE)contributes to normal skeletal muscle contractility in young but not in aged skeletal muscle.Aging 3,621-634(2011).
32.Brotto,M.&Abreu,E.L.Sarcopenia:pharmacology of today and tomorrow.J.Pharmacol.Exp.Ther.343,540-546(2012).
33.Mann,C.J.et al.Aberrant repair and fibrosis development in skeletal muscle.Skelet Muscle 1,21(2011).
34.Indra,A.K.et al.Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis:comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T)and Cre-ER(T2)recombinases.Nucleic Acids Res.27,4324-4327(1999).
35.Warming,S.,Costantino,N.,Court,D.L.,Jenkins,N.A.&Copeland,N.G.Simple and highly efficient BAC recombineering using galK selection.Nucleic Acids Res.33,e36(2005).
36.Fry,C.S.et al.Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy.FASEB J.28,1654-1665(2014).
37.Bouxsein,M.L.et al.Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.J.Bone Miner.Res.25,1468-1486(2010).
38.Zhao,X.et al.Compromised store-operated Ca2+entry in aged skeletal muscle.Aging Cell 7,561-568(2008).
39.Dobin,A.et al.STAR:ultrafast universal RNA-seq aligner.Bioinformatics 29,(15-21(2013).
40.Breese,M.R.&Liu,Y.NGSUtils:a software suite for analyzing and manipulating next-generation sequencing datasets.Bioinformatics 29,494-496(2013).
41.Liao,Y.,Smyth,G.K.&Shi,W.featureCounts:an efficient general purpose program for assigning sequence reads to genomic features.Bioinformatics 30,923-930(2014).
42.Ewels,P.,Magnusson,M.,Lundin,S.&Kaller,M.MultiQC:summarize analysis results for multiple tools and samples in a single report.Bioinformatics 32,3047-3048(2016).
43.Robinson,M.D.,McCarthy,D.J.&Smyth,G.K.edgeR:a Bioconductor package for differential expression analysis of digital gene expression data.Bioinformatics 26,139-140(2010).
44.McCarthy,D.J.,Chen,Y.&Smyth,G.K.Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation.Nucleic Acids Res.40,4288-4297(2012).
45.Mi,H.et al.PANTHER version 11:expanded annotation data from Gene Ontology and Reactome pathways,and data analysis tool enhancements.Nucl.Acids Res 45(D1),D183-D189(2017).
2.Javaheri,B.et al.Deletion of a single beta-catenin allele in osteocytes abolishes the bone anabolic response to loading.J.Bone Miner.Res.29,705-715(2014).
3.Tatsumi,S.et al.Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction.Cell.Metab.5,464-475(2007).
4.Das,S.&Sakthiswary,R.Bone metabolism and histomorphometric changes in murine models treated with sclerostin antibody:a systematic review.Curr.Drug.Targets.14,1667-1674(2013).
5.Padhi,D.,Jang,G.,Stouch,B.,Fang,L.&Posvar,E.Single-dose,placebo-controlled,randomized study of AMG 785,a sclerostin monoclonal antibody.J.Bone Miner.Res.26,19-26(2011).
6.Delmas,P.D.Clinical potential of RANKL inhibition for the management of postmenopausal osteoporosis and other metabolic bone diseases.J.Clin.Densitom.11,325-338(2008).
7.Shimada,T.&Fukumoto,S.FGF23 as a novel therapeutic target.Adv.Exp.Med.Biol.728,158-170(2012).
8.Elefteriou,F.&Yang,X.Genetic mouse models for bone studies--strengths and limitations.Bone 49,1242-1254(2011).
9.Kalajzic,I.et al.In vitro and in vivo approaches to study osteocyte biology.Bone54,296-306(2013).
10.Dallas,S.L.,Xie,Y.,Shiflett,L.A.&Ueki,Y.Mouse Cre Models for the Study of Bone Diseases.Curr.Osteoporos.Rep.16,466-477(2018).
11.Abou-Khalil,R.&Colnot,C.Cellular and molecular bases of skeletal regeneration:what can we learn from genetic mouse models?Bone 64,211-221(2014).
12.Yang,W.et al.Dentin matrix protein 1 gene cis-regulation:use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo.J.Biol.Chem.280,20680-20690(2005).
13.Lu,Y.et al.DMP1-targeted Cre expression in odontoblasts and osteocytes.J.Dent.Res.86,320-325(2007).
14.Gorski,J.P.et al.Deletion of Mbtps1(Pcsk8,S1p,Ski-1)Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age.J.Biol.Chem.291,4308-4322(2016).
15.Lim,J.,Burclaff,J.,He,G.,Mills,J.C.&Long,F.Unintended targeting of Dmp1-Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice.Bone Res.5,16049(2017).
16.Bivi,N.et al.Cell autonomous requirement of connexin 43 for osteocyte survival:consequences for endocortical resorption and periosteal bone formation.J.Bone Miner.Res.27,374-389(2012).
17.Powell,W.F.Jr et al.Targeted ablation of the PTH/PTHrP receptor in osteocytes impairs bone structure and homeostatic calcemic responses.J.Endocrinol.209,21-32(2011).
18.Brunkow,M.E.et al.Bone dysplasia sclerosteosis results from loss of the SOSTgene product,a novel cystine knot-containing protein.Am.J.Hum.Genet.68,577-589(2001).
19.Balemans,W.et al.Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein(SOST).Hum.Mol.Genet.10,537-543(2001).
20.Balemans,W.&Van Hul,W.Extracellular regulation of BMP signaling in vertebrates:a cocktail of modulators.Dev.Biol.250,231-250(2002).
21.van Bezooijen,R.L.et al.Sclerostin is an osteocyte-expressed negative regulator of bone formation,but not a classical BMP antagonist.J.Exp.Med.199,805-814(2004).
22.van Bezooijen,R.L.et al.Sclerostin in mineralized matrices and van Buchem disease.J.Dent.Res.88,569-574(2009).
23.Winkler,D.G.et al.Osteocyte control of bone formation via sclerostin,a novel BMP antagonist.EMBO J.22,6267-6276(2003).
24.Poole,K.E.et al.Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation.FASEB J.19,1842-1844(2005).
25.Xiong,J.et al.Osteocytes,not Osteoblasts or Lining Cells,are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone.PLoS One 10,e0138189(2015).
26.Amrein,K.et al.Sclerostin and its association with physical activity,age,gender,body composition,and bone mineral content in healthy adults.J.Clin.Endocrinol.Metab.97,148-154(2012).
27.M?dder,U.I.et al.Relation of age,gender,and bone mass to circulating sclerostin levels in women and men.J.Bone Miner.Res.26,373-379(2011).
28.Moester,M.J.,Papapoulos,S.E.,L?wik,C.W.&van Bezooijen,R.L.Sclerostin:current knowledge and future perspectives.Calcif.Tissue Int.87,99-107(2010).
29.Zimmermann,C.et al.Histological characterization and biochemical analysis of paraspinal muscles in neuromuscularly healthy subjects.Muscle Nerve 52,45-54(2015).
30.Lynch,G.S.,Hinkle,R.T.,Chamberlain,J.S.,Brooks,S.V.&Faulkner,J.A.Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old.J.Physiol.535(Pt 2),591-600(2001).
31.Thornton,A.M.et al.Store-operated Ca(2+)entry(SOCE)contributes to normal skeletal muscle contractility in young but not in aged skeletal muscle.Aging 3,621-634(2011).
32.Brotto,M.&Abreu,E.L.Sarcopenia:pharmacology of today and tomorrow.J.Pharmacol.Exp.Ther.343,540-546(2012).
33.Mann,C.J.et al.Aberrant repair and fibrosis development in skeletal muscle.Skelet Muscle 1,21(2011).
34.Indra,A.K.et al.Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis:comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T)and Cre-ER(T2)recombinases.Nucleic Acids Res.27,4324-4327(1999).
35.Warming,S.,Costantino,N.,Court,D.L.,Jenkins,N.A.&Copeland,N.G.Simple and highly efficient BAC recombineering using galK selection.Nucleic Acids Res.33,e36(2005).
36.Fry,C.S.et al.Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy.FASEB J.28,1654-1665(2014).
37.Bouxsein,M.L.et al.Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.J.Bone Miner.Res.25,1468-1486(2010).
38.Zhao,X.et al.Compromised store-operated Ca2+entry in aged skeletal muscle.Aging Cell 7,561-568(2008).
39.Dobin,A.et al.STAR:ultrafast universal RNA-seq aligner.Bioinformatics 29,(15-21(2013).
40.Breese,M.R.&Liu,Y.NGSUtils:a software suite for analyzing and manipulating next-generation sequencing datasets.Bioinformatics 29,494-496(2013).
41.Liao,Y.,Smyth,G.K.&Shi,W.featureCounts:an efficient general purpose program for assigning sequence reads to genomic features.Bioinformatics 30,923-930(2014).
42.Ewels,P.,Magnusson,M.,Lundin,S.&Kaller,M.MultiQC:summarize analysis results for multiple tools and samples in a single report.Bioinformatics 32,3047-3048(2016).
43.Robinson,M.D.,McCarthy,D.J.&Smyth,G.K.edgeR:a Bioconductor package for differential expression analysis of digital gene expression data.Bioinformatics 26,139-140(2010).
44.McCarthy,D.J.,Chen,Y.&Smyth,G.K.Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation.Nucleic Acids Res.40,4288-4297(2012).
45.Mi,H.et al.PANTHER version 11:expanded annotation data from Gene Ontology and Reactome pathways,and data analysis tool enhancements.Nucl.Acids Res 45(D1),D183-D189(2017).