Name: a cre-lox p recombination approach for the detection of cell fusion in vivo
Uploaded: 2012-01-04T12:00:00
Description: Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

Forfatterne vil gerne takke Dr. Peiman Hemmati (Department of Medicine, University of Wisconsin-Madison) for generøst at give H1 MSC, og Dr. Tim Hacker, Dr. Gouqing Song og Ms Jill Koch fra University of Wisconsin Cardiovascular Physiology Core facilitet for at udføre mus operationer. Dette arbejde blev støttet af National Science Foundation gennem en Graduate Research Fellowship til Brian Freeman og NIH R21 HL089679.

1. Donor Cell transfektion <ol><li> Harvest mesenchymale stamceller (MSC, der stammer fra H1 embryonale stamceller, venligst doneret af Dr. Peiman Hematti, alternativt kan enhver celle type af enhver art hypotese at sammensmelte in vivo være ansat), når 70 til 80% sammenflydende med 1X trypsin (Mediatech, Manassas VA) i 5 min. inaktivere trypsin med α-MEM komplette medium (antibiotika gratis, Invitrogen, Carlsbad CA) 18. Centrifuger ved 300 xgi 5 min. </li><li> Omhyggeligt aspir...

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Metoden beskrevet her tillader det, for første gang, diskret identifikation og tidsmæssig analyse af cellefusions i organismer, herunder små dyr. Den tilgang kombinerer Cre-LoxP rekombination med efterfølgende BioPhotonic billedanalyse. Den fremgangsmåde er modtagelig for sporing ikke kun celle-celle fusion, men også virus-celle fusion og så kunne vise sig nyttigt til sporing af virusinfektioner. Billede analyse er hurtigt og det er muligt at forestille sig mange små dyr samtidigt. Påvisning af fusion e...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Neon transfektion System </td><td> Invitrogen, Carlsbad, CA </td><td> MPK5000 </td><td></td></tr><tr><td> Neon 100 μL Kit </td><td> Invitrogen, Carlsbad, CA </td><td> MPK10025 </td><td> Indeholder R og E-buffer </td></tr><tr><td> a-MEM pulver </td><td> Invitrogen, Carlsbad, CA </td><td> 12000-022 </td><td></td></tr><tr><td> Føtalt bovint serum (FBS) </td><td> Hyclone, Logan UT </td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C-Tg (CMV-CRE) 1Cgn / J </td><td> Jackson Laboratory, Bar Harbor, ME </td><td> 006054 </td><td></td></tr><tr><td> Trypsin 10X </td><td> Fisher Scientific, Forest Lawn, NJ </td&gt; <td> MT-25 til 054-Cl </td><td></td></tr><tr><td> L-Glutamin </td><td> Fisher Scientific, Forest Lawn, NJ </td><td> 25030-081 </td><td></td></tr><tr><td> D-Luciferin </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> 122796 </td><td></td></tr><tr><td> Xenogen Biophotonic Imaging System </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> IVIS Spectrum </td><td></td></tr><tr><td> Natrium Biocarbonate </td><td> Sigma Aldrich, St. Louis, MO </td><td> S6014-500G </td><td></td></tr><tr><td> Ikke-essentielle aminosyrer </td><td> Invitrogen, Carlsbad, CA </td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

Muligheden af ​​to eller flere celler af samme type til at smelte er blevet udnyttet i metazoans hele evolutionen til at danne mange komplekse organer, herunder skelet muskler, knogler og moderkage. Moderne undersøgelser viser sammensmeltning af celler af samme type, giver forbedret funktion. For eksempel, når trofoblast cellerne i moderkagen sikringen til at danne syncytiotrophoblast den syncytiotrophoblast er bedre i stand til at transportere næringsstoffer og hormoner på tværs af maternel-føtal barriere end kondenseret trophoblasts 1-4. Nyere undersøgelser viser, sammensmeltning af celler af forskellige typer kan direkte celle skæbne. Den &quot;reversion&quot; eller ændring af celle skæbne ved fusion blev engang anset for at være begrænset til cellekultur systemer. Men fremkomsten af stamcelletransplantation førte til opdagelsen af os og andre, at stamceller kan smelte sammen med somatiske celler in vivo, og at fusion letter stamceller differentiering 5-7. Således Cellefusion er et reguleret proces, capable fremme cellernes overlevelse og differentiering og dermed kan være af central betydning for udvikling, reparation af væv og endda patogenesen af ​​sygdommen. Begrænsning studiet af cellen fusion, er mangel på passende teknologi, der 1) præcist at identificere fusion produkter og til 2) track fusion produkter over tid. Her præsenterer vi en ny tilgang til at løse både begrænsninger via induktion af bioluminescens på fusion (figur 1);. Bioluminescens kan detekteres med høj følsomhed in vivo 8-15 Vi benytter en konstruktion indkodning af ildflue luciferase (Photinus pyralis) genet placeret ved siden af et stop codon flankeret af LoxP sekvenser. Når celler, der udtrykker dette gen sikring med celler, der udtrykker Cre recombinase protein, er de LoxP steder kløvet og stop signalet er fjernet så transskription af luciferase. Fordi signalet er induciBLE, forekomsten af ​​falsk-positive signaler er meget lav. I modsætning til eksisterende metoder, som udnytter Cre / LoxP-system 16, 17, har vi indarbejdet et &quot;levende&quot; afsløring signal og dermed har råd til for første gang mulighed for at spore kinetik cellefusions in vivo. For at demonstrere den tilgang, mus allestedsnærværende udtrykker Cre recombinase tjente som modtagere af stamceller transficeret med en konstruktion til at udtrykke luciferase nedstrøms en floxed stopper codon. Stamceller er blevet transplanteret via intramyocardial injektion og efter transplantationen intravital billede analyse blev udført for at spore tilstedeværelsen af fusion produkter i hjertet og det omgivende væv over tid. Denne strategi kunne tilpasses til at analysere cellefusions i enhver vævstype på ethvert udviklingstrin, sygdom eller voksent væv reparation.

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

En metode til at spore celle fusion i levende organismer over tid er beskrevet. Den fremgangsmåde benytter Cre-<em&gt; LoxP</em&gt; Rekombination til at inducere luciferase udtryk ved celle fusion. Den selvlysende genererede signal kan påvises i levende organismer ved hjælp af BioPhotonic billeddannelse med en følsomhed på påvisning af ~ 1.000 celler i perifert væv.

En Cre-Lox P Rekombination Fremgangsmåde til påvisning af Cell Fusion In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

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A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

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