Name: genetic modification and recombination of salivary gland organ cultures
Uploaded: 2013-01-28T12:00:00
Description: Watch this Scientific Journal Video about Genetic Modification and Recombination of Salivary Gland Organ Cultures at JoVE.com

Forfatterne vil gerne takke Dr. Deirdre Nelson for personer kommentarer og for kritisk gennemlæsning af manuskriptet. Dette arbejde blev finansieret af NIH tilskud DE019244, DE019197, og DE021841 til ML, F32DE02098001 til SJS, og C06 RR015464 til universitetet i Albany, SUNY.

Protokollen indeholder fire hovedtrin, som afbildet i fig. 1. Alle trin er beskrevet i alle detaljer. Adenovirus konstruktion og viral rensning skal udføres forud for organhøst til anvendelse i den genetiske transduktion af dissekerede epiteliale ansatser. Alle standard BSL-2 sikkerhedsforanstaltninger skal følges, når man arbejder med adenovirus. 1. Muse embryonale submandibulære kirtel (SMG) Høst og mikrodissektion <ol><li> Aflive timed-drægtige mus (udavlet stamm...

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Den ex vivo epitel-mesenchymal rekombinationsteknik blev først offentliggjort for submandibulære spytkirtlerne i 1981 16. I denne protokol, udvider vi på den oprindelige metode, ved hjælp af adenovirusinfektion at manipulere epitelcelle genekspression inden for rammerne af en rekombineret kirtel. Vi viser, at en procentdel af de epiteliale celler inficeres med adenovirus, mens andelen af ​​celler, der er inficerede afhænger af egenskaberne af viral promotor, viral titer, og viral renhed. Det...

<table border="1"><tbody><tr><td> Navn på Reagent </td><td> Firma </td><td> Catalog Number </td><td> Kommentarer </td></tr><tr><td> DMEM / Ham &#39;s F12-medium uden phenolrødt </td><td> Life Technologies </td><td> 21041-025 </td><td></td></tr><tr><td> Penicillin og streptomycin </td><td> Life Technologies </td><td> 15070-163 </td><td> 10X lager </td></tr><tr><td> Dispase </td><td> Life Technologies </td><td> 17105-041 </td><td> Frys Engangskameraer alikvoter ved-20C </td></tr><tr><td> BSA </td><td> Sigma </td><td> A2934-100G </td><td> Fraktion V, lavt endotoxin </td></tr><tr><td> Adeno-X-GFP </td><td> BD Biosciences </td><td> 8138-1 </td><td> Bør være høj titer (1x10 10 pfu / ml). CsCI-rensede vira er mere effektive end spalte-oprensede virus i dette assay. </td></tr><tr><td> 16% Paraformaldehyd </td><td&gt; Electron Microscopy Sciences </td><td> 15.710 </td><td> Fortyndet til 2% i PBS med 5% sucrose (vægt / volumen) </td></tr><tr><td> 1X phosphatpufret saltvand (PBS) </td><td> Life Technologies </td><td> 70011-044 </td><td> Fremstillet ud fra 10X stock </td></tr><tr><td> Hanks balancerede saltopløsning </td><td> Life Technologies </td><td> 14175095 </td><td> ingen Calcium, ingen Magnesium, ingen phenolrødt </td></tr><tr><td> Transferrin </td><td> Sigma </td><td> T8158 </td><td> 25 mg / ml stamopløsning i DMEM/F12 media. Frys engangsbrug alikvoter ved-20C </td></tr><tr><td> L-ascorbinsyre (vitamin C) </td><td> Sigma </td><td> A4403 </td><td> 75 mg / ml stamopløsning i DMEM/F12 media.Freeze engangs-alikvoter ved -20 ° C </td></tr><tr><td></td><td></td><td></td><td> Tabel 1. Liste over reagenser, som kræves til SMG rekombination protokol. </td></tr><tr><td></td><td></td><td></td&gt; <td></td></tr><tr><td> 10 cm sterile plastskåle </td><td> Corning </td><td> 430.167 </td><td> Ikke-vævskultur-behandlede plader kan også anvendes. </td></tr><tr><td> Stereo dissektionsmikroskop med transmitteret lys base </td><td> Nikon </td><td> SMZ645 </td><td> Enhver stereo dissektionsmikroskop kan bruges, der har en lys base. </td></tr><tr><td> 35 mm vævskulturskåle </td><td> Falcon </td><td> 353.001 </td><td> Ikke-vævskultur-behandlede plader kan også anvendes. </td></tr><tr><td> 50 mm diameter mikrobrønde skåle </td><td> Mattek Corporation </td><td> P50-G-1.5-14F </td><td></td></tr><tr><td> Nuclepore Track-Etch membranfiltre </td><td> Whatman </td><td> 110.405 </td><td> 13 mm diameter, 0,1 mm porestørrelse </td></tr><tr><td> Widefield fluorescensmikroskop </td><td> Carl Zeiss, USA </td><td> Axio Observer Z1 </td><td> Enhver fluorescensmikroskop (oprejst, jegnverted eller stereo dissektionsmikroskop) kan anvendes til at overvåge GFP-ekspression ved lav forstørrelse med en vedhæftet digitalkamera. </td></tr><tr><td> Konfokalt mikroskop </td><td> Leica Microsystems </td><td> TCS SP5 </td><td> Konfokal mikroskopi er nødvendig for at se detaljerede cellestrukturer. Helst konfokalt mikroskop kan anvendes. </td></tr><tr><td> Tidsindstillet-gravide hunmus, stamme CD-1 eller ICR </td><td> Charles River Labs </td><td></td><td> Fostre høstes på dag 13 (med dag stik opdagelse udpeget som dag 0). </td></tr><tr><td> Skalpelblad # 11 </td><td> Fin Science Tools </td><td> 10.011-00 </td><td></td></tr><tr><td> Skalpel håndtag # 3 </td><td> Fin Science Tools </td><td> 10.003-12 </td><td></td></tr><tr><td> Dumont # 5 pincet inox legering, 0,05 mm X 0,02 mm </td><td> Fin Science Tools </td><td> 11.252-20 </td><td> Ideel til høst kirtler fra embryoner </td></tr><tr><td&gt; Dumont # 5 tang dumostar legering, 0.05mm X 0.01mm </td><td> Fin Science Tools </td><td> 11.295-20 </td><td> Fine tips er nødvendige for at fjerne mesenkym fra epitel. Wolfram nåle kan også anvendes. </td></tr><tr><td></td><td></td><td></td><td> Tabel 2. Udstyr, som anvendes i SMG rekombination protokol. </td></tr></tbody></table>

genetic modification and recombination of salivary gland organ cultures

Forgrening morfogenese forekommer under udviklingen af ​​mange organer, og den embryoniske muse-glandula submandibularis (SMG) er en klassisk model for studiet af forgrening morfogenese. I udviklingslandene SMG, indebærer denne proces iterative trin epithelial knop og kanal dannelse, til sidst føre til et komplekst forgrenet netværk af acini og kanaler, som tjener til at frembringe og modificere / transportere spyt, henholdsvis i mundhulen 1 - 3. Epitel-associeret basalmembran og aspekter af mesenchymal rum, herunder mesenchym-celler, vækstfaktorer og den ekstracellulære matrix, der produceres af disse celler, er afgørende for forgrening mekanisme, selv om hvordan de cellulære og molekylære begivenheder er koordineret stadig dårligt forstået 4 . Undersøgelsen af ​​de molekylære mekanismer kørsel epitelial morfogenese forskud vores forståelse af udviklingsmæssige mekanismer og giver et indblik i mulige Regenertive medicin tilgange. Sådanne undersøgelser er blevet hæmmet på grund af manglen på effektive metoder til genetisk manipulation af spyt epitel. Currently, adenoviral transduktion er den mest effektive metode til målretning af epitelceller hos voksne kirtler in vivo 5. I embryonale eksplantater, hindrer tæt mesenkym og basalmembranen omgiver epitelcellerne viral adgang til epitelcellerne. Hvis mesenkym fjernes, kan epithelet transficeres med adenovira, og epitel ansatser kan genoptage forgrening morfogenese i nærvær af Matrigel eller laminin-111 6,7. Mesenkym-free epitel Rudiment vækst kræver også yderligere supplering med opløselige vækstfaktorer og ikke fuldt rekapitulere forgrener morfogenese som den forekommer i intakte kirtler 8. Her beskriver vi en teknik, der letter adenoviral transduktion af epitelceller og kultur i den transficerede epithelium med tilhørende mesenkym. Efter mikrodissektion af embryonale SMGS, fjernelse af mesenkym, og virusinfektion af epithelet med et GFP-indeholdende adenovirus, viser vi, at epithelet spontant rekombinerer med uinficerede mesenkym, idet den gentog intakt SMG glandulær struktur og forgrenet morfogenese. Den genetisk modificerede epitelcelle population kan nemt overvåges ved anvendelse af standard fluorescensmikroskopi metoder, hvis fluorescens-mærkede adenovirale konstruktioner anvendes. Vævet rekombination her beskrevne fremgangsmåde er i øjeblikket den mest effektive og tilgængelig fremgangsmåde til transfektion af epitelceller med en vildtype-eller mutant vektor i en kompleks 3D væv konstruktion, som ikke kræver generering af transgene dyr.

Strømmen af de store eksperimentelle trin er skitseret i figur 1.. Et eksempel på en intakt SMG, en isoleret epithelial ansats, og dens tilsvarende mesenkym er vist i figur 2. Brightfield billeder af rekombinerede SMGS, som fortsat undergår forgrening morfogenese, når dyrkes ex vivo for de anførte tidspunkter, er vist i figur 3.. Rekombineret kirtler dyrket i 48 timer udtrykker epithelial GFP er vist i fig. 4. Konfokale billeder af rekombi...

Watch this Scientific Journal Video about Genetic Modification and Recombination of Salivary Gland Organ Cultures at JoVE.com

Genetic Modification and Recombination of Salivary Gland Organ Cultures

En teknik til at genmanipulere epitelceller i hele<em&gt; Ex vivo</em&gt; Dyrkede embryonale muse submandibulære kirtler (SMGS) ved hjælp af viral genoverførsel er beskrevet. Denne fremgangsmåde drager fordel af den medfødte evne SMG epitel og mesenkym spontant at rekombinere efter separation og infektion af epitelceller ansatser med adenovirale vektorer.

Genetisk modifikation og Rekombination af spytkirtel organkulturer

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

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