Зародышевых клеток и трансплантации тканей яичка Xenografting у мышей
Summary
Протоколы для половых клеток и трансплантации xenografting ткани яичка описаны. Зародышевых клеток трансплантации приводит к донорам производных сперматогенез в яичках и получатель представляет собой функциональный анализ восстановления для идентификации сперматогониальные стволовых клеток (SSC). Ткани яичка xenografting воспроизводит развитие семенников и сперматогенез различных видов донора в реципиент мышей.
Abstract
Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial β-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.
Protocol
Discussion
1. Зародышевых клеток трансплантации
Зародышевых клеток трансплантация дает только функциональный анализ для однозначного подтверждения наличия сперматогониальные стволовых клеток (SSC) в клеточной популяции. Только ГНЦ может дома и колонизировать ниши SSC на базальной ?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Работа в лаборатории авторов была поддержана Министерством сельского хозяйства США / CSREES / NRICGP (2007-35203-18213); NIH / NCRR (2 R01 RR17359-06), NIH / NIEHS (1 R21 ES014856-01A2) и Альберта инновационную – Решения здравоохранения.
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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