鸡胚胎干细胞单细胞克隆制备方法的比较

The production of single-cell clone from chicken embryonic stem cells

提取鸡X期胚盘细胞,体外培养传至第3代,采用口吸管法、细胞稀释法及克隆环法将细胞集落分离成单个细胞,并将其接种至96孔板上,每孔1个细胞,采用细胞化学法和免疫荧光法检测细胞表面标志物,探讨鸡胚胎干细胞单细胞克隆制备的实际操作的可行性,并对其生物学特性进行鉴定。结果表明:口吸管法、克隆环法、细胞稀释法克隆率分别为0、1.0%、4.2%。3种方法相比较,细胞稀释法具有操作简单易行、实验时间短、对细胞伤害小等优点,经碱性磷酸酶活性和阶段特异性表面抗原1免疫荧光鉴定均呈阳性,扩增出的克隆能稳定增殖且不分化。

Single-cells were derived from the 3rd passage multiplied colonel stem cell （ES） of stage X. Three different methods were used, cell limiting dilution method, mouth sucker method and clone link method to pick up single cell and seed into individual well of a 96-well plate, and every well contained one cell. The cell＇ s special markers by cytochemistry and histoimmunochemistry were detected. The results indicated that there was no one clone formed by mouth sucker method, 1 by clone link method and 4 by cell limiting dilution method. We concluded that cell limiting dilution method was the easiest and it took less time. Cell limiting dilution method to produce single-cell colonel can be use. The 5 clones expressed a series of surface markers, such as： alkaline phosphotase, stage-specific embryonic antigen-1.