Gene Expression In Aging-related Embryonic Development

基因表达序列分析(SAGE)提供了一个定量的、具有代表性的和全面的差异基因表达谱。我们使用SAGE分析来生成定量的成绩单评估，这已被证明比其他技术更快速、更经济。我们使用基于RT-PCR的技术来确定一些中胚层和心脏限制性基因产物在体外分化的EC来源的心肌细胞中表达的时间点。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(βMHC)在分化第三天前就有表达，但我们发现αMHC和MLC2V的明显诱导表达开始得相对较晚(αMHC和MLC2V分别为5+0.5和3+1)。GATA-4是一种参与调控心肌收缩蛋白基因表达的转录因子，在分化早期高度表达，在分化3+1.5天后，其表达水平降至成人心脏组织水平。这表明这一时间段对应于心脏分化的早期阶段。我们现在已经使用SAGE的协议来分析这些时间段内的表达序列图谱。从三个基于P19 EC细胞的文库(未分化的P19细胞，分化天数为3+0.5和3+3.0)中共测序了150,354个标签。在43,432个独特标签中，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鼠标序列都不匹配。在所研究的每个分化阶段，对转录本的时间受限的诱导或抑制是发育特征的一个共同特征，表明分化特异的过程对于心脏发育前期和早期是必要的。随着分化程度的增加，归一化标签频率分析显示357个基因产物的表达发生了显著变化(p&lt；0.01)。我们利用定量聚合酶链式反应和原位杂交来分析一些差异调控最强的转录本的时间和空间分布。其中几种产品在胎儿或成人心脏中显示出心脏优势。这项研究中确定的新基因产物为分析人类和小鼠胚胎干细胞的心脏前期和早期发育过程提供了一个框架。

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.