우리는 시약과 마우스와 Zou 실험실의 구성원에 대한 Weiguo Zou 교수와 S. 카토 교수에게 감사유용한 토론을. 우리는 또한 상하이 아홉 번째 인민 병원의 두개 안면 이상을위한 디지털화 된 Stomatology 및 연구 센터에 대한 실험실에 감사드립니다. 이 작품은 중국 국립자연과학재단(NSFC)[81570950,81870740,81800949]의 보조금으로 부분적으로 지원되었다. 상하이 서밋 & 고원 분야, 상하이 정밀 의학 연구소, 상하이 9인조 병원, 상하이 자오동 의과대학 [JC201809], 상하이 자오동 대학 의과대학 고원혁신팀 인센티브 프로젝트 상하이 제9인민병원 상하이자오퉁대학교의 학과 [JYJC201902]의 종합연구기금. L.J.는 우수 청소년 의료 인재, 상하이 "의학 재능의 떠오르는 별" 청소년 개발 프로그램 및 상하이 자오퉁 대학의 "첸 싱" 프로젝트의 학자입니다.

여기에 설명 된 동물에 관한 모든 방법은 상하이 자오퉁 의과 대학의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다.
1. 골세포 특정 Stat3 삭제 마우스의 사육
참고: Stat3fl/fl 마우스는 상업적으로 수득하였다. Ctsk-Cre 마우스는 S. 카토 (도쿄 대학, 도쿄, 일본12)에의해 제공되었다. 마우스는 표준화된 조건하에서 기관 동물 시설에서 특정 병원체가 없는 (SPF) 조건하에서 사육되고 유지되었다.
<ol><li>같은 나이의 두 여성 마우스와 성적으로 성숙한 남성 마우스를 쌍. 18 일 후, 신생아에 대한 매일 검사를 확인하십시오. 임신 한 여성 마우스를 분리하고 필요한 경우 혼자 보관하십시오. 암컷 마우스가 페어링의 1 달 이내에 임신하지 않은 경우 다른 사육 케이지 중 남성 마우스를 변경합니다.</li>
	<li>크로스 Stat3fl/fl 마우스 Ctsk-Cre 마우스 (F0). 지노티핑을 위해 꼬리를 자르고 남성 Stat3fl /+유지; Ctsk-Cre 마우스는 성적으로 성숙할 때까지 생쥐이며, 이는 약 6주(F1)입니다. 다음 프라이머사용: Stat3 F-TTGACCTGTTCCTACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Stat3 R-CCCTAGATTAGCCAGCACA; Ctsk-cre F-GAACGCACTTCGACCA; Ctsk-cre R-GCTAACCCGTTCGTTCGTTC.</li>
	<li>십자가 6 주 된 남성 Stat3fl/+; 여성 Stat3fl/fl 마우스를 가진 Ctsk-Cre 마우스. 지노티핑을 위해 꼬리를 자르고 남성 Stat3 fl/fl을유지하십시오. Ctsk-Cre 마우스는 성적으로 성숙할 때까지 생쥐이며, 이는 약 6주(F2)입니다.</li>
	<li>크로스 6 주 된 남성 Stat3 fl/fl; Ctsk-Cre 마우스 와 여성 Stat3fl/fl 마우스 (F3). 지노티핑을 위해 꼬리를 자르고 오래된 번식 마우스를 시간에 젊은 쥐로 교체하십시오 (F3 +N).</li>
</ol>2. 표본 수집
<ol><li>8주 된 남성 Stat3fl/fl 및 Stat3Ctsk 쓰레기메이트 마우스6쌍을 이산화탄소 질식으로 분리하여 안락사시합니다. 참고: CO2 유량은 분당 케이지 볼륨의 30%를 대체합니다(예: 45cm x 30cm x 30cm 케이지의 경우CO2 유량은 40L/min)입니다.</li>
	<li>마우스를 척추 위치에 놓습니다. 손으로 양자 엉덩이 관절을 부드럽게 탈바릅니다. 안과 가위를 사용하여 경골에서 수직으로 피부를 잘라낸 다음 뒷다리에서 전체 피부를 해부합니다.</li>
	<li>뒷다리를 분리하기 위해 가위로 오른쪽 엉덩이 관절과 무릎 관절의 관절 인대를 잘라냅니다. 트로챈터와 비골의 접합을 잘라낸 다음 뒷다리를 4% 파라포름알데히드에 담가 두습니다. 3단계에서 오른쪽 뒷다리를 유지합니다. 적절하게 완전히 몰입하고 4 % 파라 포름 알데히드로 골수를 고정하기 위해 양쪽 끝에서 뼈를 잘라.</li>
	<li>왼쪽 엉덩이 관절과 무릎 관절의 관절 인대를 가위로 자르고 연조직을 부드럽게 제거하고 경골과 대퇴골을 조심스럽게 분리합니다. 75%의 에탄올에 경골과 대퇴골을 따로 담급다. 단계 4에 대한 페모라를 유지하고 단계 6경골. 트로챈터를 그대로 유지하십시오.</li>
</ol>3. 파라핀 섹션 준비
<ol><li>오른쪽 뒷다리를 4°C에서 48h로 수정합니다.</li>
	<li>데칼시피: 1x PBS로 시편을 10분 3회 부드럽게 세척합니다. 15% EDTA(ddH2O의 800mL에서 150g EDTA, 10x PBS의 100mL)로 시편을 3~4주 동안 초음파 탈량제로 하여 뼈가 구부러질 수 있다. 매일 신선한 탈균 액으로 대체하십시오.</li>
	<li>1x PBS로 시편을 3배 부드럽게 씻은 다음 하룻밤 사이에 4°C에서 75% 에탄올에 담가 두십시오.</li>
	<li>탈수: 둘째 날에는 각각 1시간 씩 95% 에탄올, 100% 에탄올, 자일렌으로 시편을 순차적으로 담급니다.</li>
	<li>표본을 1/2 자일렌 1/2 파라핀에 30분 동안 담가 두어 두드셔도 됩니다. 하룻밤 사이에 65°C에서 파라핀에 표본을 담그면 됩니다.</li>
	<li>포함: 포함에 적합한 포함 탱크를 선택합니다. 경골을 균일하게 아래에 놓습니다. 대퇴골과 경골을 90° 각도로 놓습니다. 파라핀이 완전히 냉각되고 고화된 후 포함 탱크에서 제거합니다. 표본을 번호로 하룻밤 동안 -20 °C에 저장합니다.</li>
	<li>마이크로토메를 사용하여 5 μm 두께의 부분을 지속적으로 절단합니다. 20-40 섹션을 잘라. 37°C 물에 섹션을 확산하고 현미경 슬라이드에 부착하고 하룻밤 사이에 42 °C에서 구우십시오.</li>
</ol>4. 마이크로 CT 스캐닝 및 분석
<ol><li>마이크로 CT 스캐너로 왼쪽 페모라를 스캔합니다. 해상도: 10 μm; 전압 : 70 kV; 전류: 114 μA; 플리터: 0.5mm 알; 회전 단계: 0.5°.</li>
	<li>제조업체의 지시에 따라 스캐너의 지원 소프트웨어를 사용하여 피질 뼈와 좌골 뼈의 3D 이미지를 재구성합니다. ROI는 말단 성장 플레이트에 가깝고 페모라 의 중간에 피질 뼈의 총 1mm 폭에 있습니다.</li>
	<li>정량적 미세 아키텍처 매개 변수를 계산: 뼈 미네랄 밀도 (BMD), 뼈 볼륨 분획 (BV/TV), 궤적 두께 (Tb.Th.), 궤적 수 (Tb.N.), 궤적 분리 (Tb.Sp.), 및 피질 뼈 두께 (Ct.Th.).</li>
</ol>5. 트랩 염색
<ol><li>파라핀 섹션을 65°C에서 30분간 굽습니다.</li>
	<li>데왁스: 10분 동안 자일렌에 섹션을 담그세요. 신선한 자일렌으로 이 단계를 3배 수행합니다.</li>
	<li>재수화: 100% 에탄올, 95% 에탄올, 70% 에탄올, ddH2O로 섹션에 순차적으로 몰입하여 각각 5분 동안 두 번 담가 두 번.</li>
	<li>제조업체의 지시에 따라 TRAP 염색 키트를 사용하여 염색 용액을 준비하고 37°C로 따뜻하게 합니다. 참고: TRAP 염색 솔루션은 모든 분석 전에 즉시 신선하게 준비해야 합니다.</li>
	<li>각 샘플에 50-100 μL 염색 용액을 추가하고 20-30 분 동안 37 ° C 습한 챔버에 배양하십시오. 붉은 다핵 골세포가 보일 때까지 5 분마다 가벼운 현미경으로 골의 염색 상태를 확인하십시오. ddH2O로 반응을 종료합니다.</li>
	<li>30 s용 헤마톡시린 용액의 카운터스테인. 1% 암모니아 용액에 1분간 침수하여 안정적인 청색을 연출합니다. 천천히 흐르는 수돗물로 헹구는 다.</li>
	<li>중성 발삼을 사용하여 커버립으로 섹션을 마운트하고 밤새 건조하십시오.</li>
	<li>현미경으로 관심 있는 3-5 필드를 캡처합니다. 이미지 J에 의해 궤적 둘레를 분석: L1로'직선'도구를 사용하여 스케일 바(Ls)의 길이를 측정한 다음,'분할된 선'도구를 L2, 물리적 길이(Lp)로 사용하여 적혈구 둘레의 길이를 측정합니다. 3개 이상의 핵을 가진 TRAP 양성 세포의 수를 계산합니다.</li>
</ol>6. 칼신과 알리자린 레드 더블 라벨링
<ol><li>표본 제제: 0일째에 20 mg/kg 칼세인(1 mg/mL in 2% NaHCO3 솔루션)과 25 mg/kg 알리자린 레드 S(AL, 2 mg/mL in H2O)를 4일째에 주입한다. 7일째에 마우스를 희생하십시오. 경골을 조심스럽게 분리하고 48 h에 대한 4 ° C에서 4 % 파라 포름 알데히드에 고정합니다.</li>
	<li>탈수: 고정 후, 경골 3배를 PBS 1x로 부드럽게 씻으십시오. 표본을 95% 에탄올, 100% 에탄올, 자일렌으로 5분 동안 따로 담가.</li>
	<li>12시간 동안 아세톤에 표본을 담그고, 1/2 아세톤 1/2 수지에서 2시간 동안, 그리고 밤새 건조 오븐에 순수한 수지에 담가 두십시오. 참고: 수지는 제조업체의 지침에 따라 시판되는 수지(예: 812 수지 포함)에 의해 제조되었습니다.</li>
	<li>포함: 퓨어 수지를 적합한 실리카 젤 포함 탱크에 넣고 거품을 피하기 위해 표본을 부드럽게 배치합니다. 48h에 대해 60°C에서 건조 오븐에서 수지를 중합한다.</li>
	<li>회전 마이크로토메로 시편을 5 μm 두께의 섹션으로 지속적으로 절단합니다. 나머지 샘플을 실온에서 건조제로 저장합니다.</li>
	<li>75%의 알코올을 한 방울로 핀셋으로 섹션을 준수하십시오. 중립 발삼을 사용하여 커버립으로 섹션을 마운트합니다. 형광 현미경으로 적색 및 녹색 형광 라벨을 캡처합니다.</li>
	<li>두 라벨링 라인(Ir.L.Wi), 단일 라벨이 부착된 반구 둘레(sL.Pm), 이중 라벨이 부착된 관두(dL.Pm) 및 총 반구 경계(Tb.Pm)사이의 너비를 측정합니다. 미네랄 정수 속도(MAR) 및 뼈 형성 속도(BFR/BS)를 계산합니다. MAR = Ir.L.Wi/간격 일. BFR/BS= (dL.Pm±SL.Pm/2)*MAR/Tb.Pm*100%.</li>
</ol>

<ol>
	<li>Zaidi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+remodeling+in+health+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+differentiation+and+activation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+outcomes+of+osteoporotic+fractures.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Practice.+Postmenopausal+Osteoporosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre/loxP+system+for+generating+tissue-specific+knockout+mouse+models.">Cre/loxP system for generating tissue-specific knockout mouse models.</a> Nutrition reviews. 62 (6), 243-246 (2004).</li><li>Elefteriou, F., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+for+bone+studies-Strengths+and+limitations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+STAT3+in+mediating+the+cell+growth,+differentiation+and+survival+signals+relayed+through+the+IL-6+family+of+cytokine+receptors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+STAT3+signalling+in+cancer:+new+and+unexpected+biological+functions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+interleukin-6+family-mediated+Stat3+activation+in+osteoblast+differentiation+and+bone+formation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblast/osteocyte-specific+inactivation+of+Stat3+decreases+load-driven+bone+formation+and+accumulates+reactive+oxygen+species.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+controls+osteoclast+differentiation+and+bone+homeostasis+by+regulating+NFATc1+transcription.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+prevents+bone+loss+via+estrogen+receptor+alpha+and+induction+of+Fas+ligand+in+osteoclasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Cre-LoxP+system:+general+principles+to+determine+tissue-specific+roles+of+target+genes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+acid+phosphatase:+tartrate-resistant+acid+phosphatase+as+a+marker+of+osteoclast+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+machinery+for+matrix+degradation+in+bone-resorbing+osteoclasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+excision+of+a+repressive+loop+domain+in+tartrate-resistant+acid+phosphatase+by+cathepsin+K+in+osteoclasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naphthol-ASBI+phosphate+as+a+preferred+substrate+for+tartrate-resistant+acid+phosphatase+isoform+5b.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytochemistry+of+tartrate-resistant+acid+phosphatase.+Technical+considerations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+immunoassay+for+tartrate-resistant+acid+phosphatase+isoform+5b+using+the+fluorogenic+substrate+naphthol+ASBI-phosphate+and+heparin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoalkaline+phosphatase+cytochemistry.+Technical+considerations+of+endogenous+phosphatase+activity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+tartrate-resistant+Acid+phosphatase+expression+in+osteoblasts+and+osteocytes+in+experimental+osteoporosis+in+rats.">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>Tambutt&#233;, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcein+labelling+and+electrophysiology:+insights+on+coral+tissue+permeability+and+calcification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lkb1+deletion+in+periosteal+mesenchymal+progenitors+induces+osteogenic+tumors+through+mTORC1+activation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR/Raptor+signaling+is+critical+for+skeletogenesis+in+mice+through+the+regulation+of+Runx2+expression.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+demethylase+LSD1+regulates+bone+mass+by+controlling+WNT7B+and+BMP2+signaling+in+osteoblasts.">Histone demethylase LSD1 regulates bone mass by controlling WNT7B and BMP2 signaling in osteoblasts.</a> Bone Research. 6, 14 (2018).</li></ol>

저자는 공개 할 것이 없습니다.

유전자 조작 마우스 모델은 일반적으로 인간 질환의 메커니즘 및 약제 학적 치료를 연구하는 데사용된다(13). Ctsk-Cre 마우스는 골세포6의기능적 연구에 널리 사용되어 왔다. 본 연구는 골격 표현형을 분석하고 생체 내에서 골세포 활성을 제어하는 중요한 유전자를 연구하는 방법의 프로토콜을 설명했다.
조직학적 분석은 뼈 대사를 감...

골격 뼈는 인체의 주요 하중 베어링 기관이며 걷기 및 운동 중 내부 및 외부 환경 모두에서 압력을 받고있다 1. 평생 동안 뼈는 계속해서 자체 재생을 거치며, 이는 골세포와 골세포에 의해 균형을 이룹니다. 새로운 뼈를 형성하는 오래된 뼈와 골세포를 지우는 골세포의 과정은 골격 시스템2의항상성 및 기계적 기능을 유지한다. 균형에 장애가 골다공증과 같은 뼈 대사 질환을 유발할 수 있습니다. 골다공증은 과도한 골세포 활성으로 인해 전 세계적으로 널리 퍼져 있으며 사회2,3,4에상당한 경제적 손실을 초래합니다. 골다공증 치료에 사용할 수 있는 약물의 제한된 수와 부작용의 그들의 위험4에따르면, 골다세포 형성 및 활동의 세부 사항을 공개 하는 것이 중요 하다.
단핵세포/대식세포 조혈 계보로부터 유래된 골세포는 다중 핵(2~50개의 핵을 가질 수 있음)을 가지며 큰(보통 직경 100 μm 이상)2. 기전의 탐구와 골세포 질환에 대한 약물의 검열은 체외 골세포 문화를 통해 널리 개선되었지만, 복잡한 유기 반응은 표적 치료에 필수적불가결한 생체 내 증거를 만듭니다. 마우스와 인간 사이의 유전적 및 병리학적 유사성으로 인해, 유전자 조작 마우스 모델은 일반적으로 생체 내에서 인간 질환의 메커니즘 및 약제 학적 치료를 연구하는 데 사용된다6. Cre-loxP 시스템은 마우스 유전자 편집을 위해 널리 사용되는 기술이며 연구자들이 조직/세포 별 방식으로 유전자 기능을 조사할 수 있게 해 주었다5. 카테프신 K(CSTK)는 골콜라겐8을저하시킬 수 있는 골세포에 의해 분비된 시스테인 프로테아제이다. CTSK가 성숙한 골세포에서 선택적으로 표현된다는 것은 잘 받아들여진다. 따라서, Ctsk-Cre 마우스는 골세포에 대한 기능적 연구를 위한 유용한 도구로 간주되고6.
전사(STAT) 제품군의 신호 트랜스듀서 및 활성화기는 면역 및 암 진행 및 개발7,8에서고전적으로 매우 유의하다. 7개의 STAT 중 STAT3는 뼈 항상성9,10과가장 관련이 있는 것으로 보고됩니다. 여러 생체 내 연구는 골세포에서 STAT3의 특정 불활성화가 뼈 형성9,10을감소시키는 것으로 보고되었다. 그럼에도 불구 하 고, osteoclast 형성및 생체 내 뼈 대사에 STAT3의 참여에 관한 확실한 증거는 여전히 제한. 최근에는 osteoclast-특정 Stat3 삭제 마우스모델(Stat3fl/fl;)과함께 생체 내 증거를 제공했습니다. Ctsk-Cre, 이하 Stat3Ctsk)라고불리는STAT3는 골세포 분화 및 뼈대사(11)에참여한다. 본 연구에서는, 뼈 집상성에 대한 골세포별 STAT3 삭제의 영향을 연구하기 위해 Stat3Ctsk 마우스의 뼈 질량, 뼈 조직화 및 뼈 분석및 이화작용의 변화를 분석하는 데 사용한 방법과 프로토콜을 설명합니다.

<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

트랜스제닉 마우스 모델은 골세포 분화 및 활동을 제어하는 중요한 유전자를 이해하고 골다공증의 메커니즘 및 제약 치료를 연구하는 데 강력합니다. 카테핀 K (Ctsk)-Cre 마우스는 골세포의 기능적 연구에 널리 사용되어 왔다. 전사 3(STAT3)의 신호 변환기 및 활성화기는 뼈 항상성에서 관련이 있지만 생체 내의 골막세포에서의 역할은 제대로 정의되지 않습니다. STAT3가 골세포 분화 및 뼈 대사에 참여한다는 생체 내 증거를 제공하기 위해, 우리는 골세포별 Stat3 삭제 마우스모델(Stat3 fl/fl;; Ctsk-Cre)및 골격 표현형을 분석하였다. 마이크로 CT 스캐닝 및 3D 재구성은 조건부 녹아웃 마우스에서 골질량을 증가시묵화하였다. H&E 염색, 칼세인 및 알리자린 레드 더블 염색, 타르타르트 내성 산 인산인산(TRAP) 염색이 뼈 대사를 검출하기 위해 수행되었다. 요컨대, 이 프로토콜은 골격 표현형을 분석하고 생체 내에서 골세포 활성을 제어하는 중요한 유전자를 연구하기 위해 몇 가지 표준 방법과 기술을 설명합니다.

본 프로토콜을 이용하여, osteoclast 특이적 Stat3 삭제 마우스는 골세포 분화에 STAT3 삭제의 영향을 연구하기 위해 생성되었다. Stat3Ctsk 마우스와 그들의 야생형 (WT) 쓰레기는 유전자를 지운 후에 사육되고 유지되었습니다. 골수 대식세포는 골수 세포로 분리되고 배양되었고, Stat3Ctsk 마우스에서 STAT3삭제가 입증되었다(도1).
<p ...

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

이 프로토콜은 생체 내에서 골세포 활성을 제어하는 중요한 유전자를 이해하는 정식 방법을 설명합니다. 이 방법은 형질 형형과 일부 표준 기술을 사용하여 골격 표현형을 분석합니다.

조건부 Stat3 삭제 마우스 모델의 골격 표현형 분석

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

skeletal-phenotype-analysis-conditional-stat3-deletion-mouse

Universidad de Cartagena UDEC

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JoVE Journal

Biology

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

4.3K 조회수.  National Clinical Research center of Stomatology. 이 프로토콜은 생체 내에서 골세포 활성을 제어하는 중요한 유전자를 이해하는 정식 방법을 설명합니다. 이 방법은 형질 형형과 일부 표준 기술을 사용하여 골격 표현형을 분석합니다.

비디오: 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).

생성 CRISPR은 / Cas9 중재 Monoallelic 삭제할 경우는 마우스 배아 줄기 세포의 증강 기능을 연구하는

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

연구

발생학

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 치명적인 골격 돌연변이 Penetrance 자손 테스트 하 여 이동

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.

신경 근육 질환의 마우스 모델의 행동 현상형에 대한 저렴한 걸음걸이 분석

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 ...

행동분석학

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.

마우스 두개안면 조직 및 분해되지 않은 뼈의 조직 준비 및 면역 염색

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.

마우스의 간단한 임계 크기의 대퇴 결함 모델

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 ...

의학

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.

노화의 인간의 질병에 관여 유전자를 연구에 사용 된 유전자 변형 마우스에 대한 표현형 요법

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.

3차원 마이크로컴퓨터 단층촬영을 사용한 마우스의 두개악안면 기형 분석

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.

Danio rerio를 사용하여 병적 인 인간 게놈의 생체 모델링에

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

생물학

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.

결합된 공초점 및 초분해능 현미경에 의한 마우스의 신경근 접합부 특성 분석

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

신경과학

골격 기계 생물학을 탐구하기 위한 폐포골 리모델링 분석

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.

저자 스포트라이트: 폐포와 장골 리모델링을 비교하여 OTM 모델 잠재력 탐색 

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.

유도 가능한 조골 계통 특이적 Stat3 녹아웃 마우스를 사용하여 교정 치아 운동 중 치조골 리모델링 연구

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 ...