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 prof. Weiguo Zou og S. Kato for reagenser og mus og medlemmene av Zou-laboratoriet for nyttige diskusjoner. Vi takker også laboratoriet for digitalisert stomatologi og forskningssenter for kraniofaciale anomalier ved Shanghai niende folkesykehus for hjelp. Dette arbeidet ble delvis støttet av tilskudd fra National Natural Science Foundation of China (NSFC) [81570950,81870740,81800949], Shanghai Summit &Plateau Disciplines, SHIPM-mu-fondet fra Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine [JC201809], insentivprosjektet til innovasjonsteamet på høyt nivå for Shanghai Jiao Tong University School of Medicine , det tverrfaglige forskningsfondet for Shanghai niende folkesykehus, Shanghai JiaoTong University School of Medicine [JYJC201902]. Og L.J. er forsker på Outstanding Youth Medical Talents, Shanghai "Rising Stars of Medical Talent" Youth Development Program og "Chen Xing" -prosjektet fra Shanghai Jiaotong University.

Alle metoder knyttet til dyrene beskrevet her ble godkjent av Institutional Animal Care and Use Committee (IACUC) i Shanghai Jiaotong University School of Medicine.
1. Avl av osteoklastspesifikke Stat3-slettingsmus
MERK: Stat3fl/fl mus ble oppnådd kommersielt. Ctsk-Cre mus ble levert av S. Kato (University of Tokyo, Tokyo, Japan12). Musene ble oppdrettet og vedlikeholdt under spesifikke patogenfr...

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Genetisk konstruerte musemodeller brukes ofte til å studere mekanismen og farmasøytisk behandling av menneskelig sykdom13. Ctsk-Cre mus har blitt mye brukt til funksjonelle studier av osteoklaster6. Den nåværende studien beskrev metodenes protokoller for å analysere skjelettfenotype og å studere de kritiske genene som kontrollerer osteoklastaktivitet in vivo.
Histologisk analyse er den beste intuitive metoden for å oppdage beinmet...

Skjelettben er det viktigste bærende organet i menneskekroppen og er under press fra både det indre og ytre miljøet under turgåing og trening1. Gjennom ens liv går bein kontinuerlig gjennom selvfornyelse, som balanseres av osteoblaster og osteoklaster. Prosessen med osteoklaster som rydder gamle bein og osteoblaster som danner nytt bein, opprettholder homeostase og mekanisk funksjon av skjelettsystemet2. Forstyrrelser i balansen kan indusere benmetaboliske sykdommer, som osteoporose. Osteoporose, som er forårsaket av overflødig osteoklastisk aktivitet, er globalt utbredt og forårsaker betydelige økonomiske tap for samfunnet2,3,4. Ifølge det begrensede antallet legemidler som er tilgjengelige for osteoporosebehandling og deres risiko for bivirkninger4, er det viktig å avduke detaljene i osteoklastdannelse og aktivitet.
Osteoklaster avledet fra den monocytt/makrofag hematopoietiske avstamningen har flere kjerner (kan ha 2 til 50 kjerner) og er store (vanligvis større enn 100 μm i diameter)2. Selv om utforskningen av mekanismer og screening av legemidler for osteoklastiske lidelser har blitt mye forbedret via in vitro osteoklastkultur, gjør de kompliserte organiske reaksjonene in vivo-bevis uunnværlige for målrettet terapi. På grunn av genetiske og patofysiologiske likheter mellom mus og mennesker, brukes genetisk konstruerte musemodeller ofte til å studere mekanismene og de farmasøytiske behandlingene av menneskelig sykdom in vivo6. Cre-loxP-systemet er en mye brukt teknologi for musegenredigering og har gjort det mulig for forskere å undersøke genfunksjoner på en vevs- / cellespesifikk måte5. Cathepsin K (CSTK) er en cysteinprotease utskilt av osteoklaster som kan forringe ben kollagen8. Det er godt akseptert at CTSK uttrykkes selektivt i modne osteoklaster; Derfor anses Ctsk-Cre mus å være et nyttig verktøy for funksjonelle studier av osteoklaster og har blitt brukt6.
Signaltransduseren og aktivatoren av transkripsjonsfamilien (STAT) er klassisk og svært viktig i immunitet og kreftprogresjon og utvikling7,8. Blant syv STATs rapporteres STAT3 å være den mest relevante for bein homeostase9,10. Flere in vivo-studier har rapportert at spesifikk inaktivering av STAT3 i osteoblaster reduserer beindannelse9,10. Likevel er solide bevis angående deltakelse av STAT3 i osteoklastdannelse og benmetabolisme in vivo fortsatt begrenset. Nylig ga vi in vivo bevis med en osteoklast-spesifikk Stat3 sletting mus modell (Stat3fl / fl; Ctsk-Cre, heretter kalt Stat3Ctsk) at STAT3 deltar i osteoklastdifferensiering og benmetabolisme11. I den nåværende studien beskriver vi metodene og protokollene som vi brukte til å analysere endringene i beinmasse, ben histomorfologi og beinanabolisme og katabolisme av Stat3Ctsk-musene for å studere påvirkningen av osteoklastspesifikk STAT3-sletting på bein homeostase.

<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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		<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>&#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 kraftige for å forstå de kritiske genene som kontrollerer osteoklastdifferensiering og aktivitet, og for å studere mekanismer og farmasøytiske behandlinger av osteoporose. Cathepsin K (Ctsk)-Cre mus har blitt mye brukt til funksjonelle studier av osteoklaster. Signaltransduseren og aktivatoren for transkripsjon 3 (STAT3) er relevant i bein homeostase, men dens rolle i osteoklaster i vivo forblir dårlig definert. For å gi in vivo bevis på at STAT3 deltar i osteoklast differensiering og bein metabolisme, genererte vi en osteoklast-spesifikk Stat3 sletting mus modell (Stat3 fl / fl; Ctsk-Cre) og analyserte skjelettfenotypen. Mikro-CT-skanning og 3D-rekonstruksjon antydet økt benmasse i de betingede knockout-musene. H&E farging, calcein og alizarin rød dobbel farging, og tartratresistent syre fosfatase (TRAP) farging ble utført for å oppdage bein metabolisme. Kort sagt beskriver denne protokollen noen kanoniske metoder og teknikker for å analysere skjelettfenotype og for å studere de kritiske genene som kontrollerer osteoklastaktivitet i vivo.

Ved hjelp av den nåværende protokollen ble osteoklastspesifikke Stat3-slettingsmus generert for å studere påvirkningen av STAT3-sletting på osteoklastdifferensiering. Stat3Ctsk mus og deres wildtype (WT) kullkamerater ble oppdrettet og holdt etter genotyping. Benmargsmakrofager ble isolert og dyrket i osteoklaster, og STAT3-sletting i Stat3Ctsk-mus ble demonstrert (Figur 1).
Femora rekonstruk...

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 protokollen beskriver en kanonisk metode for å forstå de kritiske genene som kontrollerer osteoklastaktivitet in vivo. Denne metoden bruker en transgen musemodell og noen kanoniske teknikker for å analysere skjelettfenotype.

Skjelett fenotype analyse av en betinget stat3 sletting musemodell

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Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

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