Gene Expression In Aging-related Embryonic Development

基因表达系列分析（SAGE）产生定量的、代表性的和全面的差异基因表达谱。我们采用SAGE分析来生成定量转录本评估，该评估已被证明比其他技术更快速和经济。我们使用了RT-PCR为基础的技术，以确定的时间点，在体外分化的EC衍生的心肌细胞中表达的中胚层和心脏限制的基因产物的数量。结果表明，最早的收缩发生在分化的第5.5天（3+2.5或4+1.5）。一些收缩蛋白基因（β MHC）在分化的第三天之前表达，但我们表明α MHC和MLC 2 V的表达的明显诱导相对较晚开始（α MHC和MLC 2 V分别为5+0.5和3+1）。加塔-4是一种参与心肌收缩蛋白基因表达调控的转录因子，在分化的最早阶段高度存在，并形成3+1.5天的分化，其表达降低到成人心脏组织的水平。这表明该时间段对应于心脏分化的早期阶段。我们现在已经使用SAGE的协议来分析在这些时间段内表达的序列概况。已经从三个基于P19 EC细胞的文库（未分化的P19细胞，分化第3+0.5天和第3+3.0天）中测序了总共150，354个标签。43，432个独特标签中有10%与NR GenBank数据库中的序列匹配，而超过30%的独特标签与任何已知的小鼠序列不匹配。时间限制的诱导或抑制转录在每个分化阶段检查是一个共同的特点的发展概况，这表明分化特异性的过程是必要的前和早期心脏的发展。对分化的标准化标签频率的分析揭示了357个基因产物表达的显著变化（p<0.01）。我们已经利用定量PCR和原位杂交来分析的时间和空间分布的一些最差异调节的转录本。几种产品在胎儿或成人心脏中显示出心脏优势。本研究中鉴定的新基因产物为分析人类和小鼠胚胎干细胞的前和早期心脏发育过程提供了框架。

Serial analysis of gene expression (SAGE) yields a quantitative, representative and comprehensive differential gene expression profile. We have employed SAGE analysis to generate a quantitative transcript assessment that has proven to be much more rapid and economical than other techniques. We used an RT-PCR based technique to determine the time points where a number of mesodermal and cardiac-restricted gene products are expressed in in vitro differentiated EC derived cardiomyocytes. Results show that the earliest contractions occur on Day 5.5 of differentiation (3+2.5 or 4+1.5). Some contractile protein genes (beta MHC) are expressed before the third day of differentiation, but we show that a clear induction of expression of alpha MHC and MLC2V starts relatively later (5+0.5 and 3+1 for alpha MHC and MLC2V respectively). GATA-4, a transcription factor involved in the regulation of cardiac contractile protein gene expression is highly present in the earliest stages of differentiation, and form 3+1.5 days of differentiation, its expression is reduced to the level of adult heart tissue. This indicates that this time period corresponds to an early stage of cardiac differentiation. We have now used the protocols of SAGE to analyze expressed sequence profiles during these time periods. A total of 150,354 tags have been sequenced from three P19 EC cell-based libraries (undifferentiated P19 cells, differentiation Days 3+0.5 and 3+3.0). Ten percent of the 43,432 unique tags matched sequences in the NR GenBank database, while more than 30% of the unique tags did not match any known mouse sequence. Temporally-restricted induction or repression of transcripts at each differentiation stage examined was a common feature of the developmental profiles, suggesting that differentiation-specific processes are necessary for pre- and early cardiac development. Analysis of the normalized tag frequency with differentiation revealed significant changes (p<0.01) in expression of 357 gene products. We have utilized quantitative PCR and in situ hybridizations to analyze the temporal and spatial distribution of a number of the most differentially regulated transcripts. Several of the products have shown a cardiac predominance in either fetal or adult heart. The novel gene products identified in this study provide a framework for the analysis of pre- and early cardiac developmental processes in human and mouse embryonic stem cells.