Germ Cell Transplantation og testikel væv Xenografting i mus
Summary
Protokoller til kimcelle transplantation og testikel væv xenografting er beskrevet. Kønsceller celletransplantation resulterer i donor-afledt spermatogenesen i modtagerlandene testikler og repræsenterer et funktionelt rekonstituering analyse til identifikation af spermatogonial stamceller (SSC'er). Testis væv xenografting reproducerer testis udvikling og spermatogenese af forskellige donorarterne i recipientmus.
Abstract
Kønsceller transplantation blev udviklet af Dr. Ralph Brinster og kolleger ved University of Pennsylvania i 1994 1,2. Disse banebrydende undersøgelser viste, at mikroinjektion af kønsceller fra frugtbare donormus ind sædkanalerne af infertile modtagerlandenes mus resulterer i donor-afledte spermatogenese og sperm produktion af modtageren dyret 2. Brugen af donorhandyr bærer det bakterielle β-galactosidase-genet tillod identifikation af donor-afledte spermatogenese og transmission af donor haplotype til afkommet af modtagerlandene dyr 1. Overraskende, efter transplantation ind i lumen af de semniferøse tubuli, var transplanterede kimceller stand til at bevæge fra den luminale rum til det kælderen membranen, hvor spermatogonier er placeret 3. Det er generelt accepteret at kun SSC'er er i stand til kolonisere niche og genetablere spermatogenese i modtagerlandene testis. Derfor, kim celletransplantation tilvejebringer en funktionel tilgang at studere den stamcelle niche i de testis og at karakterisere putative spermatogonial stamceller. Til dato, bliver kimcellens transplantation anvendes til at belyse basal stamcelle biologi, til at producere transgene dyr gennem genetisk manipulation af kimceller forud for transplantation 4,5, at studere Sertoli celle-kimcelle interaktion 6,7, SSC homing og kolonisering 3, 8, såvel som SSC selvfornyelse og differentiering 9,10.
Kønsceller transplantation er også muligt i store arter 11. I disse, er de vigtigste applikationer bevarelse af fertilitet, formidling af elite genetik i dyrepopulationer, og generering af transgene dyr som studiet af spermatogenesen og SSC biologi med denne teknik er logistisk sværere og dyrere end i gnavere. Transplantation af kønsceller fra store arter ind sædkanalerne af mus resulterer i colonization af donorceller og spermatogonial ekspansion, men ikke i deres fulde differentiering formodentlig på grund af uforenelighed modtageren somatiske celler rum med kønsceller fra fylogenetisk fjerne arter 12. En alternativ metode er transplantation af kønsceller fra store arter sammen med deres omgivelser somatisk rum. Vi først rapporteret i 2002, at små fragmenter af testis væv fra umodne mænd transplanteret under ryghud immundefekte mus er i stand til at overleve og gennemgå fuld udvikling med produktion af befrugtning kompetent sæd 13. Siden da testikel væv xenografting har vist sig at være en succes i mange arter, og opstod som et værdifuldt alternativ til at studere testis udvikling og spermatogenese af store dyr i mus 14.
Protocol
Discussion
1. Kimcellens transplantation
Kønscelle celle transplantation tilvejebringer den eneste funktionelt assay for utvetydig bekræftelse af tilstedeværelsen af spermatogonial stamceller (SSC'er) i en celle population. Kun SSC'erne kan hjem til og kolonisere SSC niche på basalmembranen og igangsætte donor-afledte spermatogenese. Kønsceller transplantation gjort det muligt at studere og manipulere SSC'erne i en hidtil uset måde. Den teknik, er blevet anvendt til producere tra…
Declarações
The authors have nothing to disclose.
Acknowledgements
Arbejde fra forfatterne laboratoriet er blevet støttet af USDA / CSREES / NRICGP (2007-35203-18213), NIH / NCRR (2 R01 RR17359-06), NIH / NIEHS (1 R21 ES014856-01A2) og Alberta innoverer – Sundhed Solutions.
Materials
| Name of the reagent | Company | Catalogue number |
| Collagenase (type IV) | Sigma | C5138 |
| Trypsin-EDTA | Invitrogen | 25200-056 |
| DNaseI | Sigma | DN25 |
| DMEM | Invitrogen | 31053-028 |
| Trypan blue stain | Invitrogen | 15250-061 |
| Nylon mesh cell strainer | BD biosciences | 352340 (40μm) 352350 (70μm) |
| Busulfan | Sigma | B2635 |
| Thin-Wall Glass Capillaries | World Precision Instrument | TW 100-3 |
| BD intramedic plyethylene tubing (PE100) | BD | CA-63018-725 |
| Ethicon 6-0 Silk Suture | Ethicon | 706G |
| Wound clips | BD | 427631 |
| Sigmacote | Sigma | SL2 |
| X-gal | Sigma | B4252 |
| Potassium Ferrocyanide | Sigma | P9387 |
| Potassium Ferricyanide | Sigma | P3667 |
| magnesium chloride | Sigma | 208337 |
| sodium deoxycholate | Sigma | D6750 |
| N,N-Dimethylformamide | Sigma | D4551 |
| Igepal CA-630 | Sigma | 18896 |
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