利用巢式聚合酶链反应检测单个胚胎小体细胞中时钟基因的表达

Detection of clock genes in embryoid body derived single cells with nested RT-PCR

目的建立一组灵敏的检测时钟基因(c lock gene)的巢式聚合酶链反应(nested PCR)方法,并检测小鼠胚胎小体(EB)来源的单细胞中重要时钟基因的表达情况。方法培养小鼠胚胎干细胞(mESC),体外诱导分化为EB,提取总RNA并进行反转录,用BMAL1、CLOCK、CRY1和CRY2基因特异引物进行巢式聚合酶链反应,电泳检测扩增产物,从而建立相应的巢式PCR反应体系。利用膜片钳获取胚胎小体来源的单个细胞,通过巢式PCR反应检测相应时钟基因的表达情况。结果确定了BMAL1、CLOCK、CRY1和CRY2基因的扩增参数。证明在此条件下无非特异扩增,并且能够检测到至少两个拷贝的目的分子。利用此检测体系发现,部分胚胎小体细胞中BMAL1、CLOCK、CRY1和CRY2时钟基因环路不完整。结论巢式PCR方法能够灵敏地、而且特异地从单细胞中检测出一组时钟基因的表达,时钟基因的转录在胚胎小体细胞中并不一致,即时钟基因的环路不完整。时钟基因可能在细胞分化过程中发挥着某种作用。

Objective To establish methods for examining expression of clock genes including BMAL1 , CLOCK, CRY1 and CRY2 , and to figure out expression profile of these genes in single cell derived from embryoid body （EB）. Methods Total RNA isolated from EB was subjected to reverse transcription and amplification with clock genes specific primers to determine thermo-cycle condition, which was used consequently to examine the expression profiles of these clock genes in single cells isolated with patch clamp from embryoid bodies. Results Parameters in amplification and detection were determined. No unspecific band was amplified. At least 2 molecules could be detected with established systems. In the differentiating EB cells, co-expression of these clock genes was rare. Condusion Theses systems are sensitive enough to detect expression of clock genes in single cells. Transcription and translation loop among clock genes are not intact in differentiating EB cells, and the clock genes may play a role inthe early development and differentiation.