Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
J Cell Biochem. 2011 Jun;112(6):1514-23. doi: 10.1002/jcb.23060.
Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.
虽然禽类原始生殖细胞（PGCs）已被用于生产转基因鸟类，但它们的特性在很大程度上仍不清楚。本研究检测了鸭 EG 细胞的分离、培养、生物学特性和定向神经分化。结果表明，EG 细胞是通过从 7 日龄鸭胚中分离胚胎性腺及其周围组织获得的。与生殖腺体细胞共培养的 PGCS 生长良好。传代后，将 EG 细胞在含有细胞因子和丝裂霉素 C 的培养基中在灭活的鸭胚胎成纤维细胞（DEFs）饲养层上孵育。经过多次传代，碱性磷酸酶（ALP）和过碘酸希夫（PAS）呈阳性，细胞标志物检测 SSEA-1、SSEA-4、TRA-1-60 和 TRA-1-81 呈阳性。核型分析显示 EG 细胞保持二倍体状态，遗传特征与鸭的品种特征一致，稳定。这些细胞在 DEF 上连续生长了 11 代。在 BME、RA 和 IBMX 诱导的培养基中，EG 细胞失去未分化状态，大量神经细胞出现，并形成神经细胞网络。诱导 7 天后，甲苯胺蓝染色发现特殊的尼氏体。免疫荧光染色结果表明，分化的 EG 细胞表达巢蛋白、NSE 和 GFAP 阳性。RT-PCR 结果表明，分化的 EG 细胞表达 Nestin、NSE 和 GFAP mRNA 阳性。结果表明，RA 可明显促进鸭 EG 细胞向神经谱系的定向分化。鸭 EG 细胞将可用于生产转基因鸟类、细胞替代治疗和生殖细胞分化研究。
