Name: measuring growth and gene expression dynamics of tumor-targeted s. typhimurium bacteria
Uploaded: 2013-07-06T14:00:00
Description: Watch this Scientific Journal Video about Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria at JoVE.com

Vi takker H. Fleming for kritisk læsning og redigering af manuskriptet. Dette arbejde blev støttet af en Misrock Postdoc stipendium (TD) og NDSEG graduate stipendium (AP). SNB er en HHMI Investigator.

1.. Cell Preparation <ol><li> Passage cellelinjer under anvendelse af standard cellekulturteknikker. I dette eksperiment brugte vi OVCAR-8 celler (NCI DCTD Tumor Repository, Frederick, MD). Dyrk cellerne til et mål sammenflydning på 80 - 100%. </li><li> Cellerne inkuberes med 5 ml trypsin i 5 minutter, derefter tilsættes 5 ml RPMI-medium + FBS at inaktivere trypsin. </li><li> Harvest og tæller celler i et hæmocytometer. Resuspender i phenolrødt-frit DMEM ved en målkoncentration på 5 x 10 7 celler / ...

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Hjælp af denne procedure, er vi i stand til at generere tidsforløb for væksten og genekspression dynamik af bakterier koloniserer tumorer. Mens disse målinger rutinemæssigt udføres in vitro i batchkultur eller mikrofluidiksystemer enheder, de er meget sværere at udføre in vivo. Der er flere ændringer, der kan anvendes til disse metoder. Mens vi brugte OVCAR-8 cellelinien at generere vores musemodeller, kan en række andre cellelinier anvendes tilsvarende. For eksem...

Syntetisk biologi er skredet hastigt i det seneste årti og er nu positioneret til at påvirke vigtige problemer inden for energi og sundhed. Imidlertid har ekspansion i den kliniske arena blevet bremset af sikkerhedshensyn og et fravær af at udvikle kriterier for udformningen in vivo genetiske kredsløb. Hurtigere stor gennemslagskraft medicinske anvendelser vil kræve at udnytte metoder, interface direkte med medicinsk infrastruktur, genetiske kredsløb, der fungerer uden for det kontrollerede lab indstilling, og sikre og klinisk accepteret mikrobielle værter. En række af stammer er blevet undersøgt for cancerterapi på grund af deres evne til at vokse fortrinsvis i tumorer. Disse har omfattet C. novyi, E. coli, V. cholorae, B. longum, og S. typhimurium 3-8. S. typhimurium har skabt særlig interesse, da de har udstillet sikkerhed og tolerance i et antal humane kliniske forsøg 9-12. Disse bakterie<html> en blev oprindeligt vist at skabe antitumorvirkninger gennem stimulering af værtens immunsystem og ved udtømning af næringsstoffer, der kræves for kræft cellestofskiftet. Produktion af terapeutisk gods blev senere tilføjet gennem genetiske modifikationer. Mens disse undersøgelser udgør vigtige fremskridt i brugen af bakterier for tumor terapier, har de fleste af de eksisterende indsats påberåbt sig højt niveau udtryk, der typisk resulterer i leveringen af høje doser, off-target effekter og udvikling af værtsresistens 13-16 . Nu kan syntetisk biologi tilføje programmerbar cargo produktionen ved at udnytte beregningsmæssigt designede genetiske kredsløb, der kan udføre avancerede sensing og levering 17-20. Disse kredsløb kan være udformet til at fungere som afgivelsessystemer som registrerer tumor-specifikke stimuli og selvregulerer gods produktion efter behov. Imidlertid har studere funktionen af disse kredsløb in vivo hidtil været udfordrende. ve_content &quot;&gt; Da plasmider er de fælles rammer for syntetiske kredsløb beskriver vi en metode til at karakterisere dynamikken i plasmid-baserede genekspression in vivo ved hjælp af en musemodel. Disse metoder anvender time-lapse luminescence billedbehandling og kvantitativ måling af biofordeling. Sammen disse tilgange skabe en ramme for at studere plasmid-baserede netværk in vivo for kliniske anvendelser.

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>1 ml syringe</td>
 <td>BD Biosciences</td>
 <td>309602</td>
 <td></td>
 </tr>
 <tr>
 <td>3/10 cc Insulin Syringe</td>
 <td>BD Biosciences</td>
 <td>309301</td>
 <td></td>
 </tr>
 <tr>
 <td>PrecisionGlide Needle </td>
 <td>BD Biosciences </td>
 <td>301629</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI Medium 1640 (1X), 
 liquid, with L-glutamine</td>
 <td>Invitrogen</td>
 <td>11875-119 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sigma Aldrich</td>
 <td>A0166-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>LB Agar Broth</td>
 <td>Sigma Aldrich</td>
 <td>L2897</td>
 <td></td>
 </tr>
 <tr>
 <td>Luria-Bertani broth</td>
 <td>BD Biosciences</td>
 <td>244610</td>
 <td></td>
 </tr>
 </tbody>
</table>

measuring growth and gene expression dynamics of tumor-targeted s. typhimurium bacteria

Målet med disse eksperimenter er at generere kvantitative tidsforløb data på vækst og genekspression dynamik svækket S. typhimurium bakteriekolonier vokser inde tumorer. Vi genererede model xenotransplantattumorer i mus ved subkutan injektion af en human ovariecancer cellelinie, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). Vi forvandlet svækkede stammer af S. typhimurium bakterier (ELH430: SL1344 phoPQ-1) med et konstitutivt udtrykt luciferase (luxCDABE) plasmid til visualisering 2. Disse stammer specifikt kolonisere tumorer, mens de resterende væsentlige ikke-virulent for mus 1. Når målbare tumorer blev etableret, blev bakterier injiceret intravenøst ​​via halevenen med varierende dosering. Tumor-lokaliserede, blev bakteriel genekspression overvåges i realtid i løbet af 60 timer med enin vivo imaging-system (IVIS). På hvert tidspunkt blev tumorer udskåret, homogeniseret og udpladet til kvantificering bakteriekolonier for korrelation med genekspression data. Sammen disse data giver en kvantitativ måling af in vivo-vækst og genekspression dynamik af bakterier vokser inde tumorer.

Ved hjælp af denne protokol, er vi i stand til at generere data på in vivo vækst og genekspression dynamik tumor målrettede bakterier. Den samlede arbejdsgang er opsummeret i figur 1.. I den første fase, injicere vi bakterier (grøn) og image dyret ved hjælp IVIS at måle genekspression med luminescens (blå) som en reporter. Så i den anden fase, vi punktafgifter, blandes og plade tumorer for kolonitællinger til at bestemme antal...

Watch this Scientific Journal Video about Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria at JoVE.com

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

Måling vækst og genekspression Dynamics af Tumor-Målrettet<em&gt; S. Typhimurium</em&gt; Bakterier

Målet med disse eksperimenter er at generere kvantitative tidsforløb data på vækst og genekspression dynamik svækket<em&gt; S. typhimurium</em&gt; Bakteriekolonier vokser inde tumorer. Denne video tumorceller forberedelse og implantation, bakterier forberedelse og injektion, hel-animalsk luminescens billeddannelse, tumorexcision og bakteriekoloni tælling.

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

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Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

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