Name: differentiating functional roles of gene expression from immune and non-immune cells in mouse colitis by bone marrow transplantation
Uploaded: 2012-10-01T12:00:00
Description: Watch this Scientific Journal Video about Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation at JoVE.com

Dette arbejde blev finansieret af Pilot og gennemførlighedsundersøgelsen tilskud fra UCLA-CURE Center, Crohns og Colitis Foundation of America Career Development Award (# 2691) og National Institute of Health NIDDK K01 (DK084256) finansiering til Hon Wai Koon. Knoglemarv bestråling operation blev bistået af Bernard Levin og Scott Kitchen of UCLA Center for AIDS Research muse / humant Chimera Core facilitet. Flowcytometri operation blev bistået af UCLA Vector Core facilitet.

1. Før-du-starte Tekniske overvejelser <ol><li> Vi anbefaler brug af begge C57/BL6 vildtype-mus og knockout-mus for eksperiment, fordi mus med tilhørende cd-molekyler kan købes fra store muse leverandører. </li><li> Da det er vanskeligt at spore de vildtypemus og knockout-mus afledt donorceller efter knoglemarvstransplantation, er det nødvendigt at anvende CD45.1 mus til at repræsentere vildtypemus. (CD45.1 C57/SJL WT mus Jackson Laboratories stock # 002014). </li><li> De fleste mus kolonier er CD45.2. Vores kno...

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Denne knoglemarvstransplantation fremgangsmåde er egnet til immunologiske forskning af colitis, infektion, cancer, fedme og andre sygdomme. Denne knoglemarvstransplantation eksperiment er nødvendig, når genet af interesse udtrykkes i både knogle afledt marv og ikke-knoglemarv-afledte celler, og genet af interesse er mistænkt for at mediere sygdom af celler fra nogen af ​​populationerne. For eksempel antimikrobielle peptid cathelicidin vist sig at modulere akut colitis. Men det udtrykkes i både epithelceller og...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Cellefarvning puffer </td><td> Biolegend </td><td> # 420.201 </td></tr><tr><td> 10X RBC lyseringsbuffer </td><td> Biolegend </td><td> # 420.301 </td></tr><tr><td> FITC muse isotypekontrol </td><td> Biolegend </td><td> # 400.207 </td></tr><tr><td> PE muse-isotypekontrol </td><td> Biolegend </td><td> # 400.211 </td></tr><tr><td> PE anti-muse CD45.2 </td><td> Biolegend </td><td> # 109.807 </td></tr><tr><td> FITC anti-muse CD45.1 </td><td> Biolegend </td><td> # 110.705 </td></tr><tr><td> Anti-mus CD16/32 blokering </td><td> Biolegend </td><td> # 101.301 </td></tr><tr><td> 40 pm cell strainer </td><td> Fisherbrand </td><td> # 22363547 </td></tr><tr><td> PBS 1X </td><td&gt; MP Biomedicals </td><td> # 1860454 </td></tr><tr><td> Heparin </td><td> Fisherbrand </td><td> # BP2425 </td></tr><tr><td> Sulfatrim (SMZ) </td><td> Qualitest </td><td> NC9242720 (Fisher) </td></tr><tr><td> Lysol IC </td><td> Andwin Scientific </td><td> NC9745686 (Fisher) </td></tr><tr><td> Vaccutainer </td><td> BD </td><td> # 8000813 </td></tr><tr><td> Mus harpiksstopperen </td><td> Braintree Scientific </td><td> # TV-150 </td></tr><tr><td> Strålerøret </td><td> JL Shepherd and Associates </td><td> Mark I 68A </td></tr><tr><td> Flowcytometer </td><td> BD </td><td> BD FACSCanto II </td></tr><tr><td> Flowcytometer reagensglas </td><td> Falcon </td><td> # 352.052 </td></tr><tr><td> Digital skydelære </td><td> Fisherbrand </td><td> 14-648-17 </td></tr><tr><td> Hemoccult IKT </td><td> Beckman Coulter </td><td> G0328QW </td></tr&gt; </tbody></table>

differentiating functional roles of gene expression from immune and non-immune cells in mouse colitis by bone marrow transplantation

For at forstå den rolle, et gen i udviklingen af ​​colitis, sammenlignede vi svarene fra vildtype-mus og gen-af-interesse deficiente knockout-mus til colitis. Hvis genet af interesse udtrykkes i både knoglemarvsafledte celler og ikke-knoglemarvsafledte celler af værten, men det er muligt at skelne rollen af ​​et gen af ​​interesse i knoglemarv afledte celler og ikke-knoglemarv afledte celler ved knoglemarvstransplantation teknik. For at ændre knoglemarvsafledte celler genotype af mus, blev den oprindelige knoglemarv fra recipientmus ødelagt ved bestråling og derefter erstattes af nye donorknoglemarv af forskellige genotype. Når vildtype-mus donorknoglemarv blev transplanteret til knockout-mus, kunne vi generere knockout-mus med vildtype-genekspression i knoglemarv afledte celler. Alternativt blev da knockout mus donor knoglemarv transplanteres til vildtype-recipientmus, vildtype-mus uden gen af ​​interesse udtrykkerfra knoglemarv afledte celler blev fremstillet. Imidlertid kan knoglemarvstransplantation ikke være 100% komplet. Derfor har vi udnyttet differentieringsklynge-(CD)-molekyler (CD45.1 og CD45.2) som markører for donor-og recipientceller at spore andelen af ​​donorknoglemarv afledte celler i recipientmus og succes af knoglemarvstransplantation. Vildtypemus med CD45.1 genotypen og knockout mus med CD45.2 genotype blev anvendt. Efter bestråling af recipientmus, var donor knoglemarvceller hos forskellige genotyper infuseres ind i recipientmus. Når den nye knoglemarv regenereres at overtage sin immunitet blev musene udfordret ved kemisk middel (dextrannatriumsulfat, DSS 5%) for at inducere colitis. Vi her viste også fremgangsmåden til at inducere colitis hos mus og vurdere rollen af ​​genet af interesse udtrykkes fra knoglemarv afledte celler. Hvis genet af interesse fra knoglen afledte celler spiller en vigtig rolle i udviklingen af ​​sygdommen (f.eks colitis), kan fænotypen af ​​de recipientmus med knoglemarvstransplantation ændres væsentligt. Ved afslutningen af ​​colitis eksperimenter, afledt af knoglemarv celler i blod og knoglemarv blev mærket med antistoffer mod CD45.1 og CD45.2 og deres kvantitative forhold mellem eksistens kan anvendes til at evaluere effekten af ​​knoglemarvstransplantation ved flowcytometri. Vellykket knoglemarvstransplantation bør vise et stort flertal af donor genotype (på sigt af CD-molekyle markør) over modtager genotype i både knoglemarven og blod recipientmus.

Hvis genet af interesse spiller en vigtig rolle i immune celler under udvikling af colitis, musene modtager knoglemarv af forskellige genotype ved knoglemarvstransplantation (WT til KO eller KO til WT) bør have en ændret reaktion på DSS colitis. En af de vigtigste parametre til bestemmelse af graden af ​​colitis er H &amp; E-farvning af colon væv. Colonvæv struktur ændringer og tegn på inflammation kan kvantitativt bedømt ved H &amp; E histologi pointsystem. Kriterier for H &amp; E histologi scoring for DSS ...

Watch this Scientific Journal Video about Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation at JoVE.com

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

Knoglemarvstransplantation tilvejebringer en måde at ændre genotype af knoglemarv-afledte celler. Hvis genet af interesse udtrykkes i både knoglemarvsafledte celler og ikke-knoglemarvsafledte celler, kan knoglemarvstransplantation ændre knoglemarv afledte celler til en anden genotype uden at ændre den ikke-knoglemarvsafledte celler genotype.

Differentiering funktionelle roller af genekspression fra immun og ikke-immun celler i Mouse Colitis af knoglemarvstransplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

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