Name: a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster
Uploaded: 2013-09-11T15:15:00
Description: Watch this Scientific Journal Video about A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster at JoVE.com

Forskning i mit laboratorium er støttet af wku Biologisk Institut opstart midler, wku Research Foundation RCAP-Jeg giver # 11-8032 og et KBRIN-OMRÅDE tilskud finansieres via en forælder bevilling fra National Institute of General Medical Sciences i National Institutes Sundhedsstyrelsen under tildeling nummer 5P20GM103436-13. Jeg vil også gerne anerkende Dr. Tian Xu, i hvis laboratorium Jeg blev introduceret til denne teknik, og Dr. Raymond Pagliarini der først etableret denne teknik i Xu laboratoriet.

1.. Af godartede Ikke-invasive tumorer <ol><li> Brug aktier noteret i tabel 2 for forekomst af godartede tumorer. </li><li> Forbered en starterkultur til &quot;tester&quot; bestand af følgende genotype: y, w, ey-FLP1, Tub-Gal80, FRT40A, Act5C&gt; y +&gt; Gal4, UAS-GFP </li><li> Forbered en starterkultur til &quot;afprøvet&quot; bestand af følgende genotype: w; FRT40A, UAS-Ras V12 / CYO </li><li> Saml ti kvindelige jomfruer fra &quot;tester&quot; lager ...

<ol>
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Kræft er en kompleks sygdom med en meget bedre forståelse i dag end tidligere. Men har brug for stadig meget, der skal læres og forklares, før vi har et fuldstændigt billede af de underliggende mekanismer. Den enkle protokol præsenteres her gør det muligt at genetisk inducere benigne og invasive tumorer i en hel organisme, og derefter studere biologien forbundet med progression af tumorer i denne model. De fleste af de eksisterende teknikker i Drosophila og andre organismer enten anvende en cellekultur b...

Progression af tumorer fra en godartet til en metastatisk tilstand er en trinvis proces, der er kendetegnet ved unddragelse af beskyttende mekanismer til stede i kroppen 8. For eksempel tumorceller i kroppen skal være i stand til at unddrage sig apoptose og immunsystemet, gennembrud de specialiserede ekstracellulære matrix (ECM) kaldet basalmembran, og overvinde eventuelle sociale kontrol, der pålægges af de omkringliggende celler 8. Det er gennem et skridt klog progression at kræftcellerne erhverve evnen til at migrere og kolonisere fjerne steder i en proces, der kaldes metastaser. Vores forståelse af, hvordan tumorceller overvinder de hindringer, der følger af kroppen er stadig i sin vorden, men det spirende billedet fra forskning udført således langt point til en gentagen brug af normale udviklingsmæssige processer og signalveje, som kræftcellerne 11-13. Frugtfluen Drosophila melanogaster har bidraget enormt til vores understanding af normal udvikling og sygdom ved brug af avancerede genetiske teknikker udviklet i løbet af de sidste mange årtier 14-17. Brug mutagenese og overekspression redskaber, vi er ankommet til en bedre forståelse af forskellige onkogener og tumorsuppressorgener 18-22. Men tumormetastaser er et resultat af et samarbejde mellem flere genetiske læsioner, har primært været undersøgt i cellekulturmodeller 23,24 samt forskellige xenograftmodeller 25-27. Disse modeller selvom magtfulde har deres begrænsninger, da de ikke fuldstændig efterligne betingelserne findes i en levende organisme. Desuden transgene modeller til rådighed i mus er besværlige og ikke befordrende for genetisk analyse af invasiv adfærd 28,29. Adskillige undersøgelser har forsøgt at forstå invasion af tumorceller i Drosophila 30,31. Disse teknikker primært udnytter transplantation af primære tumorer til værter og så stole på at spore transplanted tumorer for invasion af tilstødende væv 32,33. En kraftfuld teknik kaldet MARCM 10 blev tilpasset af Pagliarini og Xu at modellere tumor invasion i Drosophila 9.. Denne elegante genetisk modellering af tumor invasion udnyttet samarbejdet mellem en aktiveret onkogen og tab af celle polaritet. Effekten af ​​denne modellering ligger i det faktum, at de invasive tumorer, er skabt i en intakt organisme derved omgå behovet for transplantation af væv. For at kunne gennemføre onkogene samarbejde, er et aktiveret onkogen som Ras V12 udtrykkes i kloner af celler i larvestadiet eye-antennal disk. Som et resultat af MARCM teknik disse kloner også er markeret med grønt fluorescerende protein (GFP) til nem visualisering og gøres homozygote for cellepolaritet mutanter som dødelig giant larver, intim og skiver store. Resultatet er GFP tagged invasive tumorer i cephalic kompleks. I denne rapport Idemonstrere, hvordan at fremkalde, og visualisere disse invasive tumorer, både i forbindelse med en intakt larver og dissekeret ud cephalic kompleks. Den tumorinduktion præsenteres her udnytter reagenser på det andet kromosom Drosophila. I tabel 2, jeg giver en liste over bestande på X og 3. kromosomer, der kan udnyttes til samme formål. Jeg tror, ​​at denne forenklede protokol vil gøre denne teknik let tilgængelige for forskere interesseret i at forstå det molekylære grundlag for tumorprogression.

<table border="1">
	<tbody>
 <tr>
 <td>10X PBS (phosphate buffered saline) pH 7.2 stock solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Chilled 1X PBS pH7.2 working solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td>Make fresh and refrigerate, can be used up to a week</td>
 </tr>
 <tr>
 <td>Flynap</td>
 <td>Carolina Biologicals</td>
 <td></td>
 <td>Fly anesthesia needed to anesthetize larvae</td>
 </tr>
 <tr>
 <td>Fixative</td>
 <td>0.1M PIPES, pH 7.2, 4% Paraformaldehyde</td>
 <td></td>
 <td>Needed to fix the dissected cephalic complex</td>
 </tr>
 <tr>
 <td>Ice Bucket</td>
 <td>Several</td>
 <td></td>
 <td>Maintain solutions on ice. Also, dissect cephalic complex in chilled 1X PBS and then place on ice in an Eppendorf tube</td>
 </tr>
 <tr>
 <td>1.7ml Eppendorf tube</td>
 <td>Various</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Glass slides, cover glass</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vectashield Mounting Media or any other mounting media</td>
 <td>Vector Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon 200 or 700 Oil</td>
 <td>Polysciences Inc. or Halocarbon.com</td>
 <td></td>
 <td>Halocarbon 200 is used to mount the larvae for visualization on a fluorescence stereoscope</td>
 </tr>
 <tr>
 <td>Sally Hansen "Hard as Nails" nail polish</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td>Needed to seal the edges of Coverslip</td>
 </tr>
 <tr>
 <td>A Leica MZ16.5 fluorescence stereomicroscope or any other fluorescence stereomicroscope</td>
 <td>Leica and others</td>
 <td></td>
 <td>Needed to observe the GFP fluorescence in larvae</td>
 </tr>
 <tr>
 <td>Dumont #5 forceps</td>
 <td>Fine Science Tools</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pyrex 9 well spot plate or any other dissection dish</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paint Brush</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Materials needed to perform the experimental protocol presented in this article.</td>
 </tr>
 </tbody>
 </table>

a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster

Drosophila har belyst vores forståelse af det genetiske grundlag for en normal udvikling og sygdom i de sidste mange årtier, og i dag fortsat bidrager enormt til vores forståelse af komplekse sygdomme 1-7. Progression af tumorer fra en godartet til en metastatisk tilstand er en kompleks begivenhed 8 og er modelleret i Drosophila for at hjælpe os til bedre at forstå det genetiske grundlag for denne sygdom 9. Her vil jeg præsentere en simpel protokol til genetisk fremkalde, observere og derefter analysere udviklingen af tumorer i Drosophila larver. Tumor induktion Teknikken er baseret på MARCM systemet 10 og udnytter samarbejdet mellem et aktiveret onkogen, Ras V12 og tab af celle polaritet gener (intim, skiver store og dødbringende giant larver) til at generere invasive tumorer 9. Jeg viser, hvordan disse tumorer kan visualiseres i intakte larver også hvordan disse kan dissekeres ud til yderligere analyse. Den forenklede protokol præsenteres her bør gøre det muligt for denne teknik til at blive udnyttet af efterforskere interesseret i at forstå den rolle, som et gen i tumor invasion.

Som et resultat af den protokol, der præsenteres her vil brugeren være i stand til at inducere godartede tumorer med overekspression af et aktiveret onkogen i larvestadiet øjet antennal imaginal disc. Brugeren vil også være i stand til at inducere invasive tumorer i øjet antennal disk ved overekspression et aktiveret onkogen i kloner af celler også mutant til cellepolaritet gen. Tumorerne let kunne visualiseres ved hjælp af en fluorescerende stereomikroskop som &quot;grønt fluorescerende&quot; væv i hele larve...

Watch this Scientific Journal Video about A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster at JoVE.com

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Samarbejde mellem en aktiveret onkogen ligesom RAS V12 og mutationer i celle polaritet gener som skreb medføre tumorvækst i Drosophila hvor tumorceller også vise invasive adfærd. Her er en simpel protokol til fremkaldelse og observation af benigne og invasive tumorer, præsenteres.

En protokol for genetiske induktion og Visualisering af godartede og invasive tumorer, i Cephalic komplekser af<em&gt; Drosophila melanogaster</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

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A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

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