Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells
Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells
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DOI：
10.2141/jpsa.0130077
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发表时间：
2014-01-01
影响因子：
1.5
通讯作者：
Kagami, Hiroshi
中科院分区：
文献类型：
--
作者：
Miyahara, Daichi;Mori, Takafumi;Kagami, Hiroshi
原始生殖细胞（Primordial germ cells，PGCs）是配子的祖细胞，其移植是获得转基因鸡的有力工具。然而，转基因整合到纯化的PGCs基因组中的频率仍然很低。建立鸡PGCs高效增殖的体外培养体系将有助于PGCs的高效转基因。在本研究中，我们优化了鸡PGCs的培养条件，以促进增殖，并评估了培养的PGCs，增殖了很长一段时间的种系传递。根据货车de Lavoir等人（2006）的方案，通过培养从2.5日龄鸡胚获得的含PGC的血液，可以获得具有与完整PGC显著相似的形态学特征的PGC样细胞（PGC-LC）。我们确定了哪些饲养细胞和哪些生长因子是改善PGC-LC增殖所必需的。雄性PGC-LC的存活和增殖在培养过程中，无论是单独的碱性成纤维细胞生长因子（bFGF）或bFGF和干细胞因子（SCF）的饲养层上的布法罗大鼠肝（BRL）细胞的基本培养基中得到增强。雄性PGC-LC可以在规定的培养条件下繁殖延长的时间。这些细胞表达生殖系特异性蛋白Vasa和未分化细胞标志物阶段特异性胚胎抗原-1（SSEA-1）以及多能性基因Nanog和PouV。培养225 d的雄性PGC-LC可向受体性腺迁移并定植于受体性腺内，移植后可传递给下一代。我们成功地产生了3个后代起源于长期培养的PGC-LC从生殖系嵌合公鸡（6%）。本研究代表了有价值的步骤，以确定一个培养条件，使PGC-LC在体外有效地繁殖长时间与维护其承诺的种系。
Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.