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 ønsker å takke dr. Peiman Hemmati (Department of Medicine, University of Wisconsin-Madison) for sjenerøst gi H1 MSCS, og Dr. Tim Hacker, Dr. Gouqing Song og Ms Jill Koch ved University of Wisconsin Cardiovascular Physiology Kjerne Facility for utføring mus surgeries. Dette arbeidet ble støttet av National Science Foundation gjennom en Graduate Research Fellowship til Brian Freeman og NIH R21 HL089679.

1. Donor Cell Transfeksjon <ol><li> Høste stamceller (MSCS, utledet fra H1 embryonale stamceller vennlig donert av Dr. Peiman Hematti, alternativt kan hvilken som helst celletype av enhver art hypothesized til sikring in vivo være ansatt) når 70 - 80% confluent med 1X trypsin (Mediatech, Manassas VA) for 5 min. inaktivere trypsin med α-MEM komplett medium (antibiotika gratis, Invitrogen, Carlsbad CA) 18. Sentrifuger ved 300 xg i 5 min. </li><li> Nøye aspirer supernatanten og r...

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	<li>Bernirschke, K. K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology of the Human Placenta. , (2000).</li><li>Hoshina, M., Boothby, M., Boime, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+localization+of+chorionic+gonadotropin+alpha+and+placental+lactogen+mRNAs+during+development+of+the+human+placenta.">Cytological localization of chorionic gonadotropin alpha and placental lactogen mRNAs during development of the human placenta.</a> J. Cell. Biol. 93, 190-198 (1982).</li><li>Johansen, M., Redman, C. W., Wilkins, T., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophoblast+deportation+in+human+pregnancy--its+relevance+for+pre-eclampsia.">Trophoblast deportation in human pregnancy--its relevance for pre-eclampsia.</a> Placenta. 20, 531-539 (1999).</li><li>Redman, C. W., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+debris,+oxidative+stress+and+pre-eclampsia.">Placental debris, oxidative stress and pre-eclampsia.</a> Placenta. 21, 597-602 (2000).</li><li>Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+fusion+of+cells+between+species+yields+transdifferentiation+and+retroviral+transfer+in+vivo.">Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo.</a> FASEB. J. 18, 548-550 (2004).</li><li>Nygren, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+hematopoietic+cells+generate+cardiomyocytes+at+a+low+frequency+through+cell+fusion,+but+not+transdifferentiation.">Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.</a> Nat. Med. 10, 494-501 (2004).</li><li>Nygren, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+and+lymphoid+contribution+to+non-haematopoietic+lineages+through+irradiation-induced+heterotypic+cell+fusion.">Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion.</a> Nat. Cell. Biol. 10, 584-592 (2008).</li><li>Kutschka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+human+BCL-2+transgene+expression+attenuates+early+donor+cell+death+after+cardiomyoblast+transplantation+into+ischemic+rat+hearts.">Adenoviral human BCL-2 transgene expression attenuates early donor cell death after cardiomyoblast transplantation into ischemic rat hearts.</a> Circulation. 114, I174-I180 (2006).</li><li>Min, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+of+cord+blood+derived+mesenchymal+stem+cell+transplantation+into+rat+myocardium.">In vivo bioluminescence imaging of cord blood derived mesenchymal stem cell transplantation into rat myocardium.</a> Ann. Nucl. Med. 20, 165-170 (2006).</li><li>Malstrom, S. E., Tornavaca, O., Meseguer, A., Purchio, A. F., West, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+characterization+and+hormonal+regulation+of+kidney+androgen-regulated+protein+(Kap)-luciferase+transgenic+mice.">The characterization and hormonal regulation of kidney androgen-regulated protein (Kap)-luciferase transgenic mice.</a> Toxicol. Sci. 79, 266-277 (2004).</li><li>Weir, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophotonic+imaging+in+HO-1.luc+transgenic+mice:+real-time+demonstration+of+gender-specific+chloroform+induced+renal+toxicity.">Biophotonic imaging in HO-1.luc transgenic mice: real-time demonstration of gender-specific chloroform induced renal toxicity.</a> Mutat. Res. 574, 67-75 (2005).</li><li>Rajashekara, G., Glover, D. A., Banai, M., O'Callaghan, D., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+bioluminescent+Brucella+melitensis+mutants+GR019+(virB4),+GR024+(galE),+and+GR026+(BMEI1090-BMEI1091)+confer+protection+in+mice.">Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice.</a> Infect. Immun. 74, 2925-2936 (1090).</li><li>Kadurugamuwa, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+biophotonic+imaging+for+monitoring+of+catheter-associated+urinary+tract+infections+and+therapy+in+mice.">Noninvasive biophotonic imaging for monitoring of catheter-associated urinary tract infections and therapy in mice.</a> Infect. Immun. 73, 3878-3887 (2005).</li><li>Ryan, P. L., Youngblood, R. C., Harvill, J., Willard, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photonic+monitoring+in+real+time+of+vascular+endothelial+growth+factor+receptor+2+gene+expression+under+relaxin-induced+conditions+in+a+novel+murine+wound+model.">Photonic monitoring in real time of vascular endothelial growth factor receptor 2 gene expression under relaxin-induced conditions in a novel murine wound model.</a> Ann. N.Y. Acad. Sci. 1041, 398-414 (2005).</li><li>Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+imaging+of+GFAP+expression+after+neuronal+damage+in+mice.">Non-invasive imaging of GFAP expression after neuronal damage in mice.</a> Neurosci. Lett. 367, 210-212 (2004).</li><li>Noiseux, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+overexpressing+Akt+dramatically+repair+infarcted+myocardium+and+improve+cardiac+function+despite+infrequent+cellular+fusion+or+differentiation.">Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation.</a> Mol. Ther. 14, 840-850 (2006).</li><li>Ajiki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+tissue+transplantation+in+rats:+fusion+of+donor+muscle+to+the+recipient+site.">Composite tissue transplantation in rats: fusion of donor muscle to the recipient site.</a> Transplant Proc. 37, 208-209 (2005).</li><li>Trivedi, P., Hematti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+immunological+characterization+of+mesenchymal+stromal+cells+from+human+embryonic+stem+cells.">Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells.</a> Exp. Hematol. 36, 350-359 (2008).</li><li>Ogle, B. M., Cascalho, M., Platt, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+implications+of+cell+fusion.">Biological implications of cell fusion.</a> Nat. Rev. Mol. Cell. Biol. 6, 567-575 (2005).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+microscopy.">ImageJ for microscopy.</a> BioTechniques. 43, 25-30 (2007).</li></ol>

Metoden er beskrevet her gjør det for første gang, diskret identifisering og tidsmessige analyse av celle fusjon i organismer, inkludert små dyr. Tilnærmingen kombinerer Cre-LoxP rekombinasjon med påfølgende Biophotonic bildeanalyse. Tilnærmingen er mottagelig for sporing ikke bare celle-celle fusjon, men også virus-cell fusion og det kan vise seg nyttig for sporing av virusinfeksjoner. Bildeanalyse er rask og det er mulig å avbilde flere små dyr samtidig. Påvisning av fusion er begrenset av hyppighet...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Neon Transfeksjon System </td><td> Invitrogen, Carlsbad, CA </td><td> MPK5000 </td><td></td></tr><tr><td> Neon 100 mL Kit </td><td> Invitrogen, Carlsbad, CA </td><td> MPK10025 </td><td> Inneholder 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> Føtal Bovine 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-25-054-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> Sodium Biocarbonate </td><td> Sigma Aldrich, St. Louis, MO </td><td> S6014-500G </td><td></td></tr><tr><td> Ikke-essensielle 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

Muligheten av to eller flere celler av samme type til sikringen har vært benyttet i metazoans gjennom evolusjonen til å danne mange komplekse organer, inkludert skjelettmuskulatur, bein og morkake. Moderne studier viser sammensmelting av celler av samme type som gir forbedret funksjon. For eksempel når trophoblast cellene i morkaken sikringen å danne syncytiotrophoblast er syncytiotrophoblast bedre i stand til å transportere næringsstoffer og hormoner over maternal-føtal barriere enn unfused trophoblasts 1-4. Nyere studier viser sammensmelting av celler av ulike typer kan direkte celle skjebne. Den &quot;hjemfall&quot; eller modifisering av celler skjebne ved fusjon en gang var tenkt å være begrenset til celle kultur systemer. Men ankomsten av stamcelletransplantasjon førte til oppdagelsen av oss og andre at stamceller kan sikringen med somatiske celler in vivo og at fusion forenkler stamcelleforskningen differensiering 5-7. Dermed er celle fusjon en regulert prosess capable fremme celle overlevelse og differensiering, og dermed kunne være av sentral betydning for utvikling, reparasjon av vev og selv de patogenesen av sykdommen. Begrense studie av celle fusion, er mangel på riktig teknologi til 1) nøyaktig identifisere fusjon produkter og til 2) spore fusion produkter over tid. Her presenterer vi en ny tilnærming for å håndtere både begrensninger via induksjon av bioluminesens ved fusjon (figur 1);. Bioluminesens kan oppdages med høy følsomhet in vivo 8-15 benytter vi en konstruksjon koding ildfluen luciferase (Photinus pyralis) genet plassert i tilknytning til en stopp kodon flankert av LoxP sekvenser. Når cellene uttrykker dette genet sikringen med celler som uttrykker Cre recombinase protein, er LoxP nettsteder kløyvde og stoppsignal er excised tillate transkripsjon av luciferase. Fordi signalet er induciBle, er forekomsten av falske positive signaler svært lav. I motsetning til eksisterende metoder som utnytter Cre / LoxP 16 system, 17, har vi innarbeidet et &quot;levende&quot; deteksjon signal og dermed råd for første gang muligheten til å spore kinetikken av celle fusjon in vivo. Å demonstrere tilnærming, mus overalt uttrykke Cre recombinase fungerte som mottakere av stamceller transfektert med en konstruksjon for å uttrykke luciferase nedstrøms av et floxed stopp kodon. Stamceller ble transplantert via intramyocardial injeksjon og etter transplantasjonen intravital bildeanalyse ble gjennomført for å spore tilstedeværelsen av fusion produkter i hjertet og omkringliggende vev over tid. Denne tilnærmingen kan tilpasses til å analysere celle fusion i alle vevstype på ethvert stadium av utvikling, sykdom eller voksen vev reparasjon.

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 for å spore celle fusion i levende organismer over tid er beskrevet. Tilnærmingen benytter Cre-<em&gt; LoxP</em&gt; Rekombinasjon å indusere luciferase uttrykk på celle fusjon. Den selvlysende signalet genereres kan påvises i levende organismer bruker Biophotonic imaging-systemer med en sensitivitet for påvisning av ~ 1000 celler i perifert vev.

En Cre-LOX P Rekombinasjon Approach for påvisning av 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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