Development of novel markers for the characterization of chicken primordial germ. cells
Development of novel markers for the characterization of chicken primordial germ. cells
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DOI：
10.1634/stemcells.2004-0208
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发表时间：
2005-05-01
期刊：
影响因子：
5.2
通讯作者：
Han, JY
中科院分区：
文献类型：
--
作者：
Jung, JS;Kim, DK;Han, JY
本研究旨在开发新的鸡原始生殖细胞（PGCs）标记，在转基因研究中具有潜在的巨大价值。从5.5日龄鸡胚中收集性腺细胞，在Dulbecco's minimal essential培养基中培养，将原代培养期间形成的PGC菌落进行三次传代培养。在播种后2小时、原代培养后（第10天）和第三代传代后（第40天），用候选标记试剂对PGCs进行鉴定。小鼠胚胎干细胞（ES）作为对照。细胞化学试剂包括周期性酸-希夫（PAS）染色剂、阶段特异性胚胎抗原抗体（SSEA-1）。SSEA-3和SSEA-4)，上皮膜抗原(EMA)-1抗体，整合素α 6和β 1抗体，几种凝集素（龙葵凝集素[STA]，芍药凝集素[DBA]，薯蓣凝集素A凝集素[Con A]和小麦胚芽凝集素[WGA]）抗体，并与SSEA-1， SSEA-3, SSEA-4，整合素α 6或整合素β 1抗体双染色，然后与凝集素STA。密度定量法鉴定pgc特异性标记物。结果表明，PAS和SSEA-1、SSEA-3、SSEA-4、EMA-1、整合素α 6和整合素β 1抗体可选择性地染色鸡PGCs。对照小鼠ES细胞与PAS、抗ssea -1和抗ema -1抗体以及整合素α 6和β 1抗体发生反应，但与SSEA-3和SSEA-4抗体无反应。鸡PGCs与凝集素STA和DBA反应，小鼠ES细胞与凝集素STA和WGA反应。三次传代的PGC菌落双染结果表明，标记试剂的同时使用没有改变染色强度。本研究表明，除了PAS和针对SSEA-1和EMA-1的抗体外，鸡PGCs的新的特异性标记还可以被凝集素STA和DBA以及针对SSEA-3和SSEA-4的抗体和整合素α 6和β 1识别。使用这些新开发的标记进行双染色可能是快速鉴定鸡PGCs的首选方法。
This study was undertaken to develop novel markers for chicken primordial germ cells (PGCs), which are of potentially enormous value in transgenic research. Gonadal cells collected from 5.5-day-old chicken embryos were cultured in a Dulbecco's minimal essential medium and the PGC colonies formed during the primary culture period were subcultured three times. Characterization of the PGCs with the candidate marker reagents was performed on the mixed cell population 2 hours after seeding, after the primary culture period (day 10), and after the third passage (day 40). Mouse embryonic stem (ES) cells were used as controls. The cytochemical reagents investigated included periodic acid-Schiff (PAS) stain, antibodies to stage-specific embryonic antigens (SSEA-1. SSEA-3, and SSEA-4), antibody to epithelial membrane antigen (EMA)-1, antibodies to integrins alpha 6 and beta 1, several lectins (Solanum tuberosum agglutinin [STA], Dolichos biflorus agglutinin [DBA], concanavalin A agglutinin [Con A],and wheat germ agglutinin [WGA]),and double staining with antibodies to SSEA-1, SSEA-3, SSEA-4, integrin alpha 6, or integrin beta 1 and then with the lectin STA. Densitometric quantification was used to identify PGC-specific markers. The results showed that chicken PGCs were stained selectively by PAS and by antibodies to SSEA-1, SSEA-3, SSEA-4, EMA-1, integrin alpha 6, and integrin beta 1. The control mouse ES cells reacted with PAS, anti-SSEA-1, and anti-EMA-1 antibodies, as well as with antibodies to integrins alpha 6 and beta 1, but not with antibodies to SSEA-3 and SSEA-4. Chicken PGCs reacted with the lectins STA and DBA, but mouse ES cells reacted with STA and WGA. The results of double staining of PGC colonies subcultured three times showed that the intensity of staining was not altered by concomitant use of the marker reagents. This study demonstrated that, in addition to PAS and antibodies to SSEA-1 and EMA-1, new specific markers of chicken PGCs are recognized by the lectins STA and DBA and by antibodies to SSEA-3 and SSEA-4 and integrins alpha 6 and beta 1. Double staining using these newly developed markers might be the method of choice for rapid characterization of chicken PGCs.