Name: germ cell transplantation and testis tissue xenografting in mice
Uploaded: 2012-02-06T12:00:00
Description: Watch this Scientific Journal Video about Germ Cell Transplantation and Testis Tissue Xenografting in Mice at JoVE.com

Arbeid fra forfattere laboratoriet har vært støttet av USDA / CSREES / NRICGP (2007-35203-18213); NIH / NCRR (2 R01 RR17359-06), NIH / NIEHS (1 R21 ES014856-01A2) og Alberta innovates - Helse Solutions.

DEL A. Germ celle transplantasjon i mus 1. Utarbeidelse av mottaker mus <ol><li> Mottakere skal være immunologisk tolerant (enten genetisk tilpasset givere eller immun-mangelfull) til donor testis cellene. </li><li> Mottakere skal være enten naturlig blottet for spermatogenesen (f.eks w / w v mus) eller utarmet av endogene kjønnsceller. Bakterie celle uttømming kan oppnås ved bestråling eller cellegifter som busulfan. I denne protokollen, beskriver vi metoden for å...

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1. Bakterie celle transplantasjon Bakterie celle transplantasjon gir den eneste funksjonelle analysen for utvetydig bekreftelse av tilstedeværelse av spermatogonial stamceller (SSCs) i en celle befolkning. Bare SSCs kan hjem til og kolonisere SSC nisje på basalmembran og initiere donor-derived spermatogenesen. Bakterie celle transplantasjon gjorde det mulig å studere og manipulere SSCs på en enestående måte. Teknikken har blitt brukt til å produsere transgene dyr gjennom genetisk manipu...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Collagenase (type IV) </td><td> Sigma </td><td> C5138 </td></tr><tr><td> Trypsin-EDTA </td><td> Invitrogen </td><td> 25200-056 </td></tr><tr><td> DNaseI </td><td> Sigma </td><td> DN25 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 31053-028 </td></tr><tr><td> Trypan blå flekken </td><td> Invitrogen </td><td> 15250-061 </td></tr><tr><td> Nylon mesh celle sil </td><td> BD Biosciences </td><td> 352340 (40μm) 352350 (70μm) </td></tr><tr><td> Busulfan </td><td> Sigma </td><td> B2635 </td></tr><tr><td> Thin-Wall Glass kapillærer </td><td> Verden presisjonsinstrument </td><td> TW 100-3 </td></tr><tr><td> BD intramedic plyethylene tubing (PE100) </td><td> BD </td><td> CA-63018-725 </td></tr><tr><td> Ethicon 6-0 Silk Suture </td><td> Ethicon </td><td> 706G </td></tr><tr><td> Sår klipp </td><td> BD </td><td> 427631 </td></tr><tr><td> Sigmacote </td><td> Sigma </td><td> SL2 </td></tr><tr><td> X-gal </td><td> Sigma </td><td> B4252 </td></tr><tr><td> Kalium Ferrocyanide </td><td> Sigma </td><td> P9387 </td></tr><tr><td> Kalium ferricyanide </td><td> Sigma </td><td> P3667 </td></tr><tr><td> magnesiumklorid </td><td> Sigma </td><td> 208337 </td></tr><tr><td> natrium deoxycholate </td><td> Sigma </td><td> D6750 </td></tr><tr><td> N, N-Dimethylformamide </td><td> Sigma </td><td> D4551 </td></tr><tr><td> Igepal CA-630 </td><td> Sigma </td><td> 18896 </td></tr></tbody></table>

germ cell transplantation and testis tissue xenografting in mice

Bakterie celle transplantasjon ble utviklet av Dr. Ralph Brinster og kolleger ved University of Pennsylvania i 1994 1,2. Disse banebrytende studier viste at mikroinjeksjon av kjønnsceller fra fruktbare donor mus inn i sædproduserende tubuli av infertile mottakerlandenes mus resulterer i donor-avledet spermatogenesen og sperm produksjon av mottakeren dyret to. Bruken av donor menn bærer bakteriell β-galaktosidase genet tillatt identifisering av donor-avledet spermatogenesen og overføring av donor haplotype til avkommet etter mottakerland dyr 1. Overraskende, etter transplantasjon inn i lumen av seminiferous tubuli, var transplanterte kjønnsceller kunne bevege seg fra luminal rommet til basalmembran hvor spermatogonier ligger tre. Det er generelt akseptert at bare SSCs er i stand til å kolonisere nisje og reetablere spermatogenesen i mottakerlandene testis. Derfor bakterie celletransplantasjon gir en funksjonell tilnærming for å studere stamcelleforskningen nisje i testis og å karakterisere antatte spermatogonial stamceller. Til dags dato er bakterie celle transplantasjon brukes til å belyse grunnleggende stamcelleforskningen biologi, for å produsere transgene dyr gjennom genetisk manipulering av kjønnsceller før transplantasjon 4,5, for å studere sertoli celle-bakterie celle interaksjon 6,7, SSC homing og kolonisering 3, 8, samt SSC selvfornyelse og differensiering 9,10. Bakterie celle transplantasjon er også mulig i store arter 11. I disse de viktigste programmene er bevaring av fruktbarhet, formidling av elite genetikk i dyrepopulasjoner, og generering av transgene dyr som studiet av spermatogenesen og SSC biologi med denne teknikken er logistikkmessig vanskeligere og dyrere enn hos gnagere. Transplantasjon av kjønnsceller fra store arter i de seminiferøse tubuli av mus resulterer i colonization av donor cellene og spermatogonial ekspansjon, men ikke i sin fulle differensiering antakelig på grunn av inkompatibilitet av mottakeren somatiske kupé med kjønnsceller fra fylogenetisk fjerne arter 12. En alternativ tilnærming er transplantasjon av kjønnsceller fra store arter sammen med sine omkringliggende somatisk avdeling. Vi først rapportert i 2002, at små fragmenter av testikkel vev fra umodne hanner transplantert under dorsal huden av immunsvikt mus er i stand til å overleve og gjennomgå full utvikling med produksjon av befruktning kompetent sperm 13. Siden da testikkel vev xenografting har vist seg å være vellykket i mange arter, og dukket opp som et verdifullt alternativ til å studere testis utvikling og spermatogenesen av store dyr i mus 14.

Watch this Scientific Journal Video about Germ Cell Transplantation and Testis Tissue Xenografting in Mice at JoVE.com

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Protokoller for bakterie celle transplantasjon og testis vev xenografting er beskrevet. Bakterie celle transplantasjon resultater i donor-derived spermatogenesen i mottakerlandene testikler og representerer en funksjonell rekonstituering analysen for identifisering av spermatogonial stamceller (SSCs). Testikkel vev xenografting reproduserer testis utvikling og spermatogenesen av ulike donor arter i mottakerlandene mus.

Germ Cell Transplantation og testis Tissue Xenografting i Mus

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

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