We thank Prof. Weiguo Zou and S. Kato for reagents and mice and the members of the Zou laboratory for useful discussions. We also thank the Laboratory for Digitized Stomatology and Research Center for Craniofacial Anomalies of Shanghai Ninth People&#39;s Hospital for assistance. This work was supported in part by grants from the National Natural Science Foundation of China (NSFC) [81570950,81870740,81800949], Shanghai Summit &#38; Plateau Disciplines, the SHIPM-mu fund from the Shanghai Institute of Precision Medicine, Shanghai Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine [JC201809], the Incentive Project of High-Level Innovation Team for Shanghai Jiao Tong University School of Medicine, the Cross-disciplinary Research Fund of Shanghai Ninth People&#39;s Hospital, Shanghai JiaoTong university School of Medicine [JYJC201902]. And L.J. is a scholar of the Outstanding Youth Medical Talents, Shanghai &#34;Rising Stars of Medical Talent&#34; Youth Development Program and the &#8220;Chen Xing&#8221; project from Shanghai Jiaotong University.

All methods relating to the animals described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiaotong University School of Medicine.
1. Breeding of osteoclast specific Stat3 deletion mice
NOTE: Stat3fl/fl mice were obtained commercially. Ctsk-Cre mice were provided by S. Kato (University of Tokyo, Tokyo, Japan12). The mice were bred and maintained under specific pathogen-free (SPF) conditions in the institutional animal facility under standardized conditions.
<ol>
	<li>Pair a sexually mature male mouse with two female mice of the same age. After 18 days, check daily checks for newborns. Separate the pregnant female mice and keep it alone if needed. Change male mice among different breeding cages if the female mice were not pregnant within 1 month of the pairing.</li>
	<li>Cross Stat3fl/fl mice to Ctsk-Cre mice (F0). Clip the tails for genotyping and keep the male Stat3fl/+; Ctsk-Cre mice until they are sexually mature, which is around 6 weeks of age (F1). Use the following primers: Stat3 F-TTGACCTGTGCTCCTACAAAAA; Stat3 R-CCCTAGATTAGGCCAGCACA; Ctsk-cre F-GAACGCACTGATTTCGACCA; Ctsk-cre R-GCTAACCAGCGTTTTCGTTC.</li>
	<li>Cross 6-week-old male Stat3fl/+; Ctsk-Cre mice with female Stat3fl/fl mice. Clip the tails for genotyping and keep the male Stat3 fl/fl; Ctsk-Cre mice until they are sexually mature, which is around 6 weeks of age (F2).</li>
	<li>Cross 6-week-old male Stat3 fl/fl; Ctsk-Cre mice with female Stat3fl/fl mice (F3). Clip the tails for genotyping and replace the old breeding mice with younger mice in time (F3+N).</li>
</ol>
2. Specimen collection
<ol>
	<li>Euthanize six pairs of 8-week old male Stat3fl/fl and Stat3Ctsk littermate mice separately with carbon dioxide asphyxiation. 
	NOTE: The CO2 flow rate displaces 30% of the cage volume per minute (e.g., for 45 cm x 30 cm x 30 cm cage the CO2 flow rate is 40 L/min).</li>
	<li>Place the mice in a supine position. Dislocate the bilateral hip joints gently by hand. Use ophthalmic scissors to cut off the skin vertically from the distal tibia and then dissect the entire skin from the hind limb.</li>
	<li>Cut off the articular ligament of the right hip joint and knee joint with scissors to separate the hind limb. Cut the trochanter and the junction of the fibula and then immerse the hind limb in 4% paraformaldehyde. Keep the right hind limbs for step 3. Appropriately cut the bone at both ends to fully immerse and fix the bone marrow with 4% paraformaldehyde.</li>
	<li>Cut the articular ligament of the left hip joint and knee joint with scissors, gently remove the soft tissue, and carefully separate the tibia and femur. Immerse the tibia and femur separately in 75% ethanol. Keep the femora for step 4 and tibiae for step 6. Ensure to keep the trochanter intact.</li>
</ol>
3. Paraffin section preparation
<ol>
	<li>Fix the right hind limb in 4% paraformaldehyde at 4 &#176;C for 48 h.</li>
	<li>Decalcify: Gently wash the specimens with 1x PBS for 10 min 3 times. Decalcify the specimens in 15% EDTA (150 g EDTA in 800 mL of ddH2O and 100 mL of 10x PBS) with an ultrasonic decalcifier for 3 to 4 weeks until the bones can be bent. Replace with fresh decalcifying fluid every other day.</li>
	<li>Gently wash the specimens 3x with 1x PBS and then immerse them in 75% ethanol at 4 &#176;C overnight.</li>
	<li>Dehydrate: On the second day, sequentially immerse specimens in 95% ethanol, 100% ethanol, and xylene, each for 1 h twice.</li>
	<li>Immerse specimens in 1/2 xylene 1/2 paraffin for 30 min. Immerse the specimens in paraffin at 65 &#176;C overnight.</li>
	<li>Embed: Select a suitable embedding tank for embedding. Place the tibia uniformly underneath. Place the femur and tibia at a 90&#176; angle. After the paraffin has fully cooled and solidified, remove it from the embedding tank. Number the specimens and store them at -20 &#176;C overnight.</li>
	<li>Cut 5 &#956;m thick sections continuously using the microtome. Cut 20&#8211;40 sections. Spread the sections on 37 &#176;C water, adhere them to microscope slides, and bake at 42 &#176;C overnight.</li>
</ol>
4. Micro-CT scanning and analysis
<ol>
	<li>Scan the left femora with a micro-CT scanner. Resolution: 10 &#956;m; Voltage: 70 kV; Current: 114 &#956;A; Fliter: 0.5 mm Al; Rotation step: 0.5&#176;.</li>
	<li>Reconstruct 3D images of the cortical bone and trabecular bone using the scanner&#8217;s supporting software following manufacturer&#8217;s instruction. ROIs are in a total 1 mm width of trabecular bone close to the distal growth plate and in a total 1 mm wide section of cortical bone in the middle of the femora.</li>
	<li>Calculate the quantitative microarchitecture parameters: bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th.), trabecular number (Tb.N.), trabecular separation (Tb.Sp.), and cortical bone thickness (Ct.Th.).</li>
</ol>
5. TRAP staining
<ol>
	<li>Bake the paraffin sections at 65 &#176;C for 30 min.</li>
	<li>Dewax: Immerse the sections in xylene for 10 min. Perform this step 3x with fresh xylene.</li>
	<li>Rehydrate: Immerse the sections sequentially in 100% ethanol, 95% ethanol, 70% ethanol, and ddH2O, each for 5 min twice.</li>
	<li>Prepare the staining solution using the TRAP staining kit following the manufacturer&#8217;s instructions and warm to 37 &#176;C. 
	NOTE: TRAP staining solution should be freshly prepared immediately before every assay.</li>
	<li>Add 50&#8211;100 &#956;L staining solution to each sample and incubate in a 37 &#176;C humid chamber for 20&#8211;30 min. Check the staining status of the osteoclasts under a light microscope every 5 min until red multinucleated osteoclasts can be seen. End the reaction with ddH2O.</li>
	<li>Counterstain in hematoxylin solution for 30 s. Create a stable blue color by immersion in 1% ammonia solution for 1 min. Rinse in slowly running tap water.</li>
	<li>Mount the sections with coverslips using neutral balsam and dry overnight.</li>
	<li>Capture 3&#8211;5 fields of interest by a microscope. Analyze the trabecular perimeter by Image J: measure the length of scale bar (Ls) using the &#8216;straight line&#8217; tool as L1, then measure the length of trabecular perimeter using the &#8216;segmented line&#8217; tool as L2, the physical length (Lp)= Ls*L2 /L1). Count the number of TRAP-positive cells with more than three nuclei.</li>
</ol>
6. Calcein and alizarin red double labeling
<ol>
	<li>Specimen preparation: Intraperitoneally inject 20 mg/kg calcein (1 mg/mL in 2% NaHCO3 solution) on day 0, and 25 mg/kg alizarin red S (AL, 2 mg/mL in H2O) on day 4. Sacrifice mice on day 7. Carefully disassociate the tibiae and fix in 4% paraformaldehyde at 4 &#176;C for 48 h.</li>
	<li>Dehydrate: After fixation, gently wash the tibiae 3x with 1x PBS. Sequentially immerse the specimens in 95% ethanol, 100% ethanol, and xylene for 5 min twice separately.</li>
	<li>Immerse the specimens in acetone for 12 h, in 1/2 acetone 1/2 resin for 2 h, and in pure resin in a drying oven overnight. 
	NOTE: The resin was prepared by commercially available resin (e.g., Embed 812 Resin) according to the manufacturer&#8217;s instructions.</li>
	<li>Embed: Add pure resin into a suitable silica gel embedding tank and place the specimens gently to avoid bubbles. Polymerize the resin in a drying oven at 60 &#176;C for 48 h.</li>
	<li>Cut the specimens into 5 &#956;m thick sections continuously with a rotary microtome. Store the rest of the samples with desiccant at room temperature.</li>
	<li>Adhere the sections with tweezers in a drop of 75% alcohol. Mount the sections with coverslips using neutral balsam. Capture red and green fluorescence labeling with a fluorescence microscope.</li>
	<li>Measure the width between two labeling lines (Ir.L.Wi), single-labeled trabecular perimeter (sL.Pm), double-labeled trabecular perimeter (dL.Pm), and total trabecular perimeter (Tb.Pm). Calculate the mineral apposition rate (MAR) and bone formation rate (BFR/BS). MAR = Ir.L.Wi/interval days. BFR/BS= (dL.Pm&#177;sL.Pm/2)*MAR/Tb.Pm*100 %.</li>
</ol>

<ol><li>Zaidi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+remodeling+in+health+and+disease%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Skeletal remodeling in health and disease.</a> Nature Medicine. 13 (7), 791-801 (2007).</li>
<li>Boyle, W. J., Simonet, W. S., Lacey, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteoclast+differentiation+and+activation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Osteoclast differentiation and activation.</a> Nature. 423 (6937), 337-342 (2003).</li>
<li>Cummings, S. R., Melton, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+and+outcomes+of+osteoporotic+fractures%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Epidemiology and outcomes of osteoporotic fractures.</a> Lancet. 359 (9319), 1761-1767 (2002).</li>
<li>Black, D. M., Rosen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+Practice.+Postmenopausal+Osteoporosis%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Clinical Practice. Postmenopausal Osteoporosis.</a> New England Journal of Medicine. 374 (3), 254-262 (2016).</li>
<li>Kos, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cre%2FloxP+system+for+generating+tissue-specific+knockout+mouse+models%5BTitle%5D)+AND+%22Nutrition+reviews%22%5BJournal%5D)">Cre/loxP system for generating tissue-specific knockout mouse models.</a> Nutrition reviews. 62 (6), Pt 1 243-246 (2004).</li>
<li>Elefteriou, F., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+mouse+models+for+bone+studies-Strengths+and+limitations%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Genetic mouse models for bone studies-Strengths and limitations.</a> Bone. 49 (6), 1242-1254 (2011).</li>
<li>Hirano, T., Ishihara, K., Hibi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roles+of+STAT3+in+mediating+the+cell+growth%2C+differentiation+and+survival+signals+relayed+through+the+IL-6+family+of+cytokine+receptors%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors.</a> Oncogene. 19 (21), 2548-2556 (2000).</li>
<li>Yu, H., Lee, H., Herrmann, A., Buettner, R., Jove, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revisiting+STAT3+signalling+in+cancer%3A+new+and+unexpected+biological+functions%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Revisiting STAT3 signalling in cancer: new and unexpected biological functions.</a> Nature Reviews Cancer. 14 (11), 736-746 (2014).</li>
<li>Itoh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+critical+role+for+interleukin-6+family-mediated+Stat3+activation+in+osteoblast+differentiation+and+bone+formation%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">A critical role for interleukin-6 family-mediated Stat3 activation in osteoblast differentiation and bone formation.</a> Bone. 39 (3), 505-512 (2006).</li>
<li>Zhou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteoblast%2Fosteocyte-specific+inactivation+of+Stat3+decreases+load-driven+bone+formation+and+accumulates+reactive+oxygen+species%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Osteoblast/osteocyte-specific inactivation of Stat3 decreases load-driven bone formation and accumulates reactive oxygen species.</a> Bone. 49 (3), 404-411 (2011).</li>
<li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STAT3+controls+osteoclast+differentiation+and+bone+homeostasis+by+regulating+NFATc1+transcription%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">STAT3 controls osteoclast differentiation and bone homeostasis by regulating NFATc1 transcription.</a> Journal of Biological Chemistry. 294 (42), 15395-15407 (2019).</li>
<li>Nakamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogen+prevents+bone+loss+via+estrogen+receptor+alpha+and+induction+of+Fas+ligand+in+osteoclasts%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts.</a> Cell. 130 (5), 811-823 (2007).</li>
<li>Kim, H., Kim, M., Im, S. K., Fang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+Cre-LoxP+system%3A+general+principles+to+determine+tissue-specific+roles+of+target+genes%5BTitle%5D)+AND+%22Laboratory+animal+research%22%5BJournal%5D)">Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes.</a> Laboratory animal research. 34 (4), 147-159 (2018).</li>
<li>Minkin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone+acid+phosphatase%3A+tartrate-resistant+acid+phosphatase+as+a+marker+of+osteoclast+function%5BTitle%5D)+AND+%22Calcified+Tissue+International%22%5BJournal%5D)">Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function.</a> Calcified Tissue International. 34 (3), 285-290 (1982).</li>
<li>Vaaraniemi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+machinery+for+matrix+degradation+in+bone-resorbing+osteoclasts%5BTitle%5D)+AND+%22Journal+of+Bone+and+Mineral+Research%22%5BJournal%5D)">Intracellular machinery for matrix degradation in bone-resorbing osteoclasts.</a> Journal of Bone and Mineral Research. 19 (9), 1432-1440 (2004).</li>
<li>Ljusberg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteolytic+excision+of+a+repressive+loop+domain+in+tartrate-resistant+acid+phosphatase+by+cathepsin+K+in+osteoclasts%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Proteolytic excision of a repressive loop domain in tartrate-resistant acid phosphatase by cathepsin K in osteoclasts.</a> The Journal of Biological Chemistry. 280 (31), 28370-28381 (2005).</li>
<li>Janckila, A. J., Takahashi, K., Sun, S. Z., Yam, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Naphthol-ASBI+phosphate+as+a+preferred+substrate+for+tartrate-resistant+acid+phosphatase+isoform+5b%5BTitle%5D)+AND+%22Journal+of+Bone+and+Mineral+Research%22%5BJournal%5D)">Naphthol-ASBI phosphate as a preferred substrate for tartrate-resistant acid phosphatase isoform 5b.</a> Journal of Bone and Mineral Research. 16 (4), 788-793 (2001).</li>
<li>Janckila, A. J., Li, C. Y., Lam, K. W., Yam, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cytochemistry+of+tartrate-resistant+acid+phosphatase.+Technical+considerations%5BTitle%5D)+AND+%22American+Journal+of+Clinical+Pathology%22%5BJournal%5D)">The cytochemistry of tartrate-resistant acid phosphatase. Technical considerations.</a> American Journal of Clinical Pathology. 70 (1), 45-55 (1978).</li>
<li>Janckila, A. J., Simons, R. M., Yam, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+immunoassay+for+tartrate-resistant+acid+phosphatase+isoform+5b+using+the+fluorogenic+substrate+naphthol+ASBI-phosphate+and+heparin%5BTitle%5D)+AND+%22Clinica+Chimica+Acta%3A+International+Journal+of+Clinical+Chemistry%22%5BJournal%5D)">Alternative immunoassay for tartrate-resistant acid phosphatase isoform 5b using the fluorogenic substrate naphthol ASBI-phosphate and heparin.</a> Clinica Chimica Acta: International Journal of Clinical Chemistry. 347 (1-2), 157-167 (2004).</li>
<li>Janckila, A. J., Yam, L. T., Li, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunoalkaline+phosphatase+cytochemistry.+Technical+considerations+of+endogenous+phosphatase+activity%5BTitle%5D)+AND+%22American+Journal+of+Clinical+Pathology%22%5BJournal%5D)">Immunoalkaline phosphatase cytochemistry. Technical considerations of endogenous phosphatase activity.</a> American Journal of Clinical Pathology. 84 (4), 476-480 (1985).</li>
<li>Solberg, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+tartrate-resistant+Acid+phosphatase+expression+in+osteoblasts+and+osteocytes+in+experimental+osteoporosis+in+rats%5BTitle%5D)+AND+%22Calcified+Tissue+International%22%5BJournal%5D)">Increased tartrate-resistant Acid phosphatase expression in osteoblasts and osteocytes in experimental osteoporosis in rats.</a> Calcified Tissue International. 94 (5), 510-521 (2014).</li>
<li>Tambutté, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcein+labelling+and+electrophysiology%3A+insights+on+coral+tissue+permeability+and+calcification%5BTitle%5D)+AND+%22Proceedings.+Biological+Sciences%22%5BJournal%5D)">Calcein labelling and electrophysiology: insights on coral tissue permeability and calcification.</a> Proceedings. Biological Sciences. 279 (1726), 19-27 (2012).</li>
<li>Han, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lkb1+deletion+in+periosteal+mesenchymal+progenitors+induces+osteogenic+tumors+through+mTORC1+activation%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Lkb1 deletion in periosteal mesenchymal progenitors induces osteogenic tumors through mTORC1 activation.</a> Journal of Clinical Investigation. 130 (5), 1895-1909 (2019).</li>
<li>Dai, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mTOR%2FRaptor+signaling+is+critical+for+skeletogenesis+in+mice+through+the+regulation+of+Runx2+expression%5BTitle%5D)+AND+%22Cell+Death+and+Differentiation%22%5BJournal%5D)">mTOR/Raptor signaling is critical for skeletogenesis in mice through the regulation of Runx2 expression.</a> Cell Death and Differentiation. 24 (11), 1886-1899 (2017).</li>
<li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+demethylase+LSD1+regulates+bone+mass+by+controlling+WNT7B+and+BMP2+signaling+in+osteoblasts%5BTitle%5D)+AND+%22Bone+Research%22%5BJournal%5D)">Histone demethylase LSD1 regulates bone mass by controlling WNT7B and BMP2 signaling in osteoblasts.</a> Bone Research. 6, 14(2018).</li>
</ol>

The authors have nothing to disclose.

Genetically engineered mouse models are commonly used for studying the mechanism and pharmaceutical treatment of human disease13. Ctsk-Cre mice have been widely used for functional studies of osteoclasts6. The present study described the protocols of the methods to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.
Histological analysis is the best intuitive method to detect bone metabolis...

Skeletal bone is the main load-bearing organ of the human body and is under pressure from both the internal and external environment during walking and exercise1. Throughout one&#8217;s life, bones continuously go through self-renewal, which is balanced by osteoblasts and osteoclasts. The process of osteoclasts clearing old bones and osteoblasts forming new bone maintains the homeostasis and mechanical function of the skeletal system2. Disturbance in the balance may induce bone metabolic diseases, such as osteoporosis. Osteoporosis, which is caused by excess osteoclastic activity, is globally prevalent and causes substantial economic losses to society2,3,4. According to the limited number of drugs available for osteoporosis treatment and their risk of adverse effects4, it is important to unveil the details of osteoclast formation and activity.
Osteoclasts derived from the monocyte/macrophage hematopoietic lineage have multiple nuclei (may have 2 to 50 nuclei) and are large (usually greater than 100 &#956;m in diameter)2. Although the exploration of mechanisms and the screening of drugs for osteoclastic disorders have been widely improved via in vitro osteoclast culture, the complicated organic reactions make in vivo evidence indispensable for the targeted therapy. Due to genetic and pathophysiological similarities between mice and humans, genetically engineered mouse models are commonly used for studying the mechanisms and the pharmaceutical treatments of human disease in vivo6. The Cre-loxP system is a widely-used technology for mouse gene editing and has enabled researchers to investigate gene functions in a tissue-/cell-specific manner5. Cathepsin K (CSTK) is a cysteine protease secreted by osteoclasts that can degrade bone collagen8. It is well accepted that CTSK is selectively expressed in mature osteoclasts; therefore, Ctsk-Cre mice are considered to be a useful tool for functional studies of osteoclasts and has been used6.
The signal transducer and activator of transcription (STAT) family is classical and highly significant in immunity and cancer progression and development7,8. Among seven STATs, STAT3 is reported to be the most relevant to bone homeostasis9,10. Several in vivo studies have reported that specific inactivation of STAT3 in osteoblasts decreases bone formation9,10. Nevertheless, solid evidence regarding the participation of STAT3 in osteoclast formation and bone metabolism in vivo is still limited. Recently, we provided in vivo evidence with an osteoclast-specific Stat3 deletion mouse model (Stat3fl/fl; Ctsk-Cre, hereafter called Stat3Ctsk) that STAT3 participates in osteoclast differentiation and bone metabolism11. In the present study, we describe the methods and protocols that we used to analyze the changes in bone mass, bone histomorphology, and bone anabolism and catabolism of the Stat3Ctsk mice in order to study the influence of osteoclast-specific STAT3 deletion on bone homeostasis.

<table><tbody><tr><td>4% Paraformaldehyde solution</td><td>Sangon biotech Co., Ltd.</td><td>E672002</td><td></td></tr><tr><td>Acetone</td><td>Shanghai Experimental Reagent Co., Ltd.</td><td>80000360</td><td></td></tr><tr><td>Alizarin</td><td>Sigma-Aldrich</td><td>A5533</td><td></td></tr><tr><td>Ammonia solution</td><td>Shanghai Experimental Reagent Co., Ltd.</td><td></td><td></td></tr><tr><td>Calcein</td><td>Sigma-Aldrich</td><td>C0875</td><td></td></tr><tr><td>&lt;em&gt;Ctsk-Cre&lt;/em&gt; mice</td><td></td><td></td><td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td></tr><tr><td>DDSA</td><td>Electron Microscopy Sciences</td><td>13710</td><td></td></tr><tr><td>DeCa RapidlyDecalcifier</td><td>Pro-Cure</td><td>DX1100</td><td></td></tr><tr><td>DMP-30</td><td>Electron Microscopy Sciences</td><td>13600</td><td></td></tr><tr><td>EDTA</td><td>Shanghai Experimental Reagent Co., Ltd.</td><td>60-00-4</td><td></td></tr><tr><td>EMBED 812 RESIN</td><td>Electron Microscopy Sciences</td><td>14900</td><td></td></tr><tr><td>fluorescence microscope</td><td>Olympus</td><td>IX73</td><td></td></tr><tr><td>Hematoxylin solution</td><td>Beyotime Biotechanology</td><td>C0107</td><td></td></tr><tr><td>Micro-CT</td><td>Scanco Medical AG</td><td>&amp;mu;CT 80</td><td></td></tr><tr><td>NaHCO3</td><td>Shanghai Experimental Reagent Co., Ltd.</td><td>10018918</td><td></td></tr><tr><td>Neutral balsam</td><td>Sangon biotech Co., Ltd.</td><td>E675007</td><td></td></tr><tr><td>NMA</td><td>Electron Microscopy Sciences</td><td>19000</td><td></td></tr><tr><td>Paraffin</td><td>Sangon biotech Co., Ltd.</td><td>A601889</td><td></td></tr><tr><td>rotary microtome</td><td>Leica</td><td>RM2265</td><td></td></tr><tr><td>&lt;em&gt;Stat3&lt;sup&gt;fl/fl&lt;/sup&gt;&lt;/em&gt; mice</td><td>GemPharmatech Co., Ltd</td><td>D000527</td><td></td></tr><tr><td>TRAP staining kit</td><td>Sigma-Aldrich</td><td>387A</td><td></td></tr><tr><td>xylene</td><td>Shanghai Experimental Reagent Co., Ltd.</td><td>1330-20-7</td><td></td></tr></tbody></table>

skeletal phenotype analysis of a conditional stat3 deletion mouse model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&#38;E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Using the present protocol, osteoclast specific Stat3 deletion mice were generated to study the influence of STAT3 deletion on osteoclast differentiation. Stat3Ctsk mice and their wildtype (WT) littermates were bred and kept after genotyping. Bone marrow macrophages were isolated and cultured into osteoclasts, and STAT3 deletion in Stat3Ctsk mice was demonstrated (Figure 1).
Femora reconstruction a...

Watch this Scientific Journal Video about Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model at JoVE.com

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

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Research

JoVE Journal

Biology

4.4K Views.  National Clinical Research center of Stomatology. Dit protocol beschrijft een canonieke methode om de kritische genen te begrijpen die de osteoclastenactiviteit in vivo controleren. Deze methode maakt gebruik van een transgeen muismodel en enkele canonieke technieken om skeletfenotype te analyseren.

Video: Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Developmental Biology

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

Behavior

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Medicine

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

Neuroscience

Analyzing Alveolar Bone Remodeling to Explore Skeletal Mechanical Biology

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial process of alveolar bone remodeling in response to mechanical force, but the underlying mechanism remains elusive. Previous studies have been unable to reveal the precise mechanism of bone remodeling in any time and space due to animal model-related restrictions. The signal transducer and activator of transcription 3 (STAT3) is important in bone metabolism, but its role in osteoblasts during OTM is unclear. To provide in vivo evidence that STAT3 participates in OTM at specific time points and in particular cells during OTM, we generated a tamoxifen-inducible osteoblast lineage-specific Stat3 knockout mouse model, applied orthodontic force, and analyzed the alveolar bone phenotype. 
Micro-computed tomography (Micro-CT) and stereo microscopy were used to access OTM distance. Histological analysis selected the area located within three roots of the first molar (M1) in the cross-section of the maxillary bone as the region of interest (ROI) to evaluate the metabolic activity of osteoblasts and osteoclasts, indicating the effect of orthodontic force on alveolar bone. In short, we provide a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describe methods for analyzing alveolar bone remodeling during OTM, thus shedding new light on skeletal mechanical biology.

Author Spotlight: Comparing Alveolar and Long Bone Remodeling to Explore OTM Model Potential 

This study provides a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describes methods for analyzing alveolar bone remodeling during orthodontic tooth movement, thus shedding light on skeletal mechanical biology.

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial ...

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

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