Name: skeletal phenotype analysis of a conditional stat3 deletion mouse model
Uploaded: 2020-07-03T14:30:00
Description: Watch this Scientific Journal Video about Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model at JoVE.com

Vi takker professor Weiguo Zou og S. Kato for reagenser og mus og medlemmerne af Zou-laboratoriet for nyttige diskussioner. Vi takker også Laboratoriet for Digitaliseret Stomatologi og Research Center for Craniofacial Anomalies of Shanghai Ninth People's Hospital for hjælp. Dette arbejde blev delvist støttet af tilskud fra National Natural Science Foundation of China (NSFC) [81570950,81870740,81800949], Shanghai Summit &Plateau Disciplines, SHIPM-mu-fonden fra Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine [JC201809], incitamentsprojektet for innovationsteam på højt niveau for Shanghai Jiao Tong University School of Medicine , den tværfaglige forskningsfond for Shanghai Ninth People's Hospital, Shanghai JiaoTong university School of Medicine [JYJC201902]. Og LJ er en forsker i Outstanding Youth Medical Talents, Shanghai "Rising Stars of Medical Talent" Youth Development Program og "Chen Xing" projekt fra Shanghai Jiaotong University.

Alle metoder vedrørende de dyr, der er beskrevet her, blev godkendt af Institutional Animal Care and Use Committee (IACUC) i Shanghai Jiaotong University School of Medicine.
1. Opdræt af osteoklastspecifikke Stat3-sletningsmus
BEMÆRK: Stat3fl/fl mus blev fremstillet kommercielt. Ctsk-Cre mus blev leveret af S. Kato (University of Tokyo, Tokyo, Japan12). Musene blev opdrættet og vedligeholdt un...

<ol>
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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. 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Gensplejsede musemodeller bruges ofte til at studere mekanismen og farmaceutisk behandling af sygdomme hos mennesker13. Ctsk-Cre mus har været meget anvendt til funktionelle undersøgelser af osteoklaster6. Denne undersøgelse beskrev protokollerne for metoderne til at analysere skeletphoenotype og til at studere de kritiske gener, der styrer osteoklastaktivitet in vivo.
Histologiske analyser er den bedste intuitive metode til at opdage...

Skeletben er det vigtigste bærende organ i menneskekroppen og er under pres fra både det indre og ydre miljø under gang og motion1. Gennem ens liv gennemgår knogler kontinuerligt selvfornyelse, som afbalanceres af osteoblaster og osteoklaster. Processen med osteoklaster, der rydder gamle knogler og osteoblaster, der danner nye knogler, opretholder skeletsystemets homøostase og mekaniske funktion2. Forstyrrelser i balancen kan fremkalde knoglemetaboliske sygdomme, såsom osteoporose. Osteoporose, som er forårsaget af overskydende osteoklastisk aktivitet, er globalt udbredt og forårsager betydelige økonomiske tab for samfundet2,3,4. Ifølge det begrænsede antal lægemidler til rådighed for osteoporose behandling og deres risiko for bivirkninger4, er det vigtigt at afsløre detaljerne i osteoklast dannelse og aktivitet.
Osteoklaster afledt af den monocytiske/makrofaglige hæmatopoietiske afstamning har flere kerner (kan have 2 til 50 kerner) og er store (normalt over 100 μm i diameter)2. Selv om udforskningen af mekanismer og screeningen af lægemidler mod osteoklastiske lidelser er blevet væsentligt forbedret via in vitro osteoklastkulturen, gør de komplicerede organiske reaktioner in vivo-beviser uundværlige for den målrettede behandling. På grund af genetiske og patofysiologiske ligheder mellem mus og mennesker anvendes genetisk manipulerede musemodeller almindeligvis til at studere mekanismerne og de farmaceutiske behandlinger af sygdomme hos mennesker in vivo6. Cre-loxP-systemet er en udbredt teknologi til musegenredigering og har gjort det muligt for forskere at undersøge genfunktioner på en vævs-/cellespecifik måde5. Cathepsin K (CSTK) er en cystein protease udskilles af osteoklaster, der kan nedbryde knogle kollagen8. Det er velkendt, at CTSK er selektivt udtrykt i modne osteoklaster; derfor anses Ctsk-Cre mus for at være et nyttigt værktøj til funktionelle undersøgelser af osteoklaster og er blevet brugt6.
Signaltransduceren og aktivatoren af transskriptionsfamilien (STAT) er klassisk og meget vigtig i immunitet og kræftudvikling og udvikling7,8. Blandt syv STAT'er rapporteres STAT3 at være den mest relevante for knoglehomøostase9,10. Flere in vivo-undersøgelser har rapporteret , at specifik inaktivering af STAT3 i osteoblaster reducerer knogledannelsen9,10. Ikke desto mindre er solid dokumentation for STAT3's deltagelse i osteoklastdannelse og knoglemetabolisme in vivo stadig begrænset. For nylig har vi givet in vivo beviser med en osteoklast-specifik stat3 sletning mus model(Stat3fl / fl; Ctsk-Cre, i det følgende benævnt Stat3Ctsk), at STAT3 deltager i osteoklast differentiering og knoglemetabolisme11. I denne undersøgelse beskriver vi de metoder og protokoller, som vi brugte til at analysere ændringerne i knoglemasse, knogle histomorphologi og knogleanabolisme og katabolisme hos Stat3Ctsk-musene for at studere indflydelsen af osteoklastspecifik STAT3-sletning på knoglehomøostase.

<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>Ctsk-Cre 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>
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			<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>&#956;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>Stat3fl/fl 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

Transgene musemodeller er stærke til at forstå de kritiske gener, der styrer osteoklastdifferentiering og aktivitet, og til at studere mekanismer og farmaceutiske behandlinger af osteoporose. Cathepsin K (Ctsk)-Cre mus har været meget anvendt til funktionelle undersøgelser af osteoklaster. Signaltransduceren og aktivatoren af transskription 3 (STAT3) er relevant i knoglehomøostase, men dens rolle i osteoklaster in vivo er stadig dårligt defineret. For at fremlægge in vivo-dokumentation for, at STAT3 deltager i osteoklastdifferentiering og knoglemetabolisme, genererede vi en osteoklastspecifik Stat3-sletningsmusmodel (Stat3 fl/fl; Ctsk-Cre) og analyserede dens skelet fænotype. Micro-CT-scanning og 3D-rekonstruktion indebar øget knoglemasse i de betingede knockoutmus. H &E farvning, calcein og alizarin rød dobbelt farvning, og tartrat-resistente syre fosfatase (TRAP) farvning blev udført for at opdage knogle metabolisme. Kort sagt beskriver denne protokol nogle kanoniske metoder og teknikker til at analysere skeletphoenotype og studere de kritiske gener, der styrer osteoklastaktivitet in vivo.

Ved hjælp af denne protokol blev osteoklastspecifikke Stat3-sletningsmus genereret for at studere INDFLYDELSEn af STAT3-sletning på osteoklastdifferentiering. Stat3Ctsk mus og deres wildtype (WT) littermates blev opdrættet og holdt efter genotyping. Knoglemarvsmakrofager blev isoleret og dyrket i osteoklaster, og STAT3-sletning i Stat3Ctsk-mus blev påvist (Figur 1).
Femora rekonstruktion og kv...

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

Denne protokol beskriver en kanonisk metode til at forstå de kritiske gener, der styrer osteoklastaktivitet in vivo. Denne metode bruger en transgen mus model og nogle kanoniske teknikker til at analysere skelet fænotype.

Skelet phenotype analyse af en betinget Stat3 Sletning Mouse Model

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Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

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