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Biologie

生殖细胞的移植和睾丸组织在小鼠异种移植

Published: February 06, 2012
doi:
10.3791/3545
, ,
1Department of Comparative Biology and Experimental Medicine,University of Calgary

Summary

生殖细胞移植和睾丸组织异种移植的协议描述。在捐助源性受援国睾丸精子的生殖细胞移植的结果和鉴定精原干细胞（SSCS）为代表的功能重建法。睾丸组织异种移植在受体小鼠睾丸发育和精子各捐助国物种再现。

Abstract

生殖细胞移植是由拉尔夫Brinster博士和他的同事在美国宾夕法尼亚大学在1994年1,2。这些开创性的研究表明，捐助衍生的精子和精子生产由收件人动物2到不孕受体小鼠的结果曲细精管生精细胞注射从肥沃的供体小鼠。的使用捐赠受援国动物携带细菌β-半乳糖苷酶基因，鉴定捐助衍生的精子和捐赠单体的传输后代男性。令人惊讶的是，进入曲细精管的管腔移植后，移植的生殖细胞能够从管腔车厢移动其中精原细胞位于基底膜。它被普遍接受，唯一的SSC能够殖民的利基，并在收件人睾丸重新建立精子。因此，生殖细胞移植提供了一个功能的方法来研究在睾丸干细胞小生境的特点推测的精原干细胞。到今天为止，生殖细胞移植用于澄清基本的干细胞生物学，生殖细胞的遗传操作，通过生产转基因动物移植4,5之前，研究Sertoli细胞的生殖细胞相互作用6,7，SSC的归巢和定植3， 8，以及SSC的自我更新和分化9,10。

11大物种生殖细胞移植也是可行的。主要应用在这些保存生育能力，传播精英在动物种群遗传学，转基因动物精子和SSC生物学研究这一技术是后勤更加困难和昂贵的，比在啮齿类动物中的一代。从大型物种的生殖细胞移植到小鼠结果在colonizat曲细精管离子的供体细胞和精原干细胞扩张，但没有充分分化，大概是由于收件人的体细胞与生殖细胞系统发育遥远的物种12间不相容。另一种方法是从大型物种与周围的体车厢的生殖细胞的移植。我们首次报道于2002年，即从下背部皮肤移植免疫缺陷小鼠的不成熟男性睾丸组织的小碎片能够生存和接受的全面发展与生产的施肥主管精子13。此后睾丸组织异种移植已被证明是成功在许多物种中，并作为一种有价值的替代研究小鼠14大型动物的睾丸发育和精子出现。

Protocol

A部分胚芽细胞移植在小鼠 1。受体小鼠的制备受助人应是免疫耐受（无论是捐助者或免疫缺陷的基因相匹配的）捐助睾丸细胞。 收件人应当要么自然没有精子（ 如 W / 宽 V小鼠）或内源性的生殖细胞耗尽。可以通过辐射或化疗药物，如马利兰生殖细胞耗竭。在这个协议中，我们描述的准备与马利兰受体小鼠的方法。 对待受助人的年龄…

Discussion

1。生殖细胞移植

生殖细胞移植提供了明确确认的精原干细胞存在于细胞的人口（SSCS）的唯一功能检测。只有共享服务中心可以回家和拓殖在基底膜SSC的利基，并发起捐助源性精子。生殖细胞移植使人们有可能以前所未有的方式SSC的研究和操纵。该技术已用于生产转基因动物通过SSCS 4的基因操纵澄清定植3,8南南合作的模式，效率和动力学研究信号通路调节SSC的?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

从作者实验室的工作已经支持农业部/ CSREES / NRICGP（2007-35203-18213）; NIH / NCRR（R01 RR17359-06），美国国立卫生研究院/ NIEHS的（1 R21的ES014856-01A2）和艾伯塔创新 – 健康解决方案。

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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References

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Tags

Germ Cell TransplantationMiceDr. Ralph BrinsterUniversity Of PennsylvaniaMicroinjectionSeminiferous TubulesSpermatogenesisSperm ProductionDonor-derivedBacterial β-galactosidase GeneOffspringLumen Of The Seminiferous TubulesBasement MembraneSpermatogoniaSSCsStem Cell NichePutative Spermatogonial Stem CellsTransgenic AnimalsGenetic ManipulationSertoli Cell-germ Cell InteractionSSC Homing And ColonizationSelf-renewal And Differentiation
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Tang, L., Rodriguez-Sosa, J. R., Dobrinski, I. Germ Cell Transplantation and Testis Tissue Xenografting in Mice. J. Vis. Exp. (60), e3545, doi:10.3791/3545 (2012).
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