Stanton J-A L, Macgregor A B, Mason C, Dameh M, Green D P L
Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand.
Mol Hum Reprod. 2007 Oct;13(10):713-20. doi: 10.1093/molehr/gam050. Epub 2007 Sep 5.
To understand early mammalian development there is a need to compare profiles of gene expression from different stages of the preimplantation mouse embryo. We describe here a method that uses gene expression data held in the UniGene database of the National Institutes of Health (NIH). The full mouse UniGene database (build #151) contains 43,104 gene clusters generated from approximately 4.1 million sequences. The Expressed Sequence Tags (EST) used to build UniGene are derived from cDNA libraries that are archived separately in the database of Expressed Sequence Tags (dbEST) database, with their own catalogue numbers. The mouse dbEST database contains 32 non-normalized dbEST libraries constructed from preimplantation stages (unfertilized oocyte, fertilized oocyte, 2-, 4-, 8- and 16-cell embryo and blastocyst). These libraries contain 219,852 EST sequences mapping to 15,731 UniGene clusters. We have developed a computational pipeline approach that imports and aggregates inventories of gene expression contained in these dbEST libraries. It uses these data to build an annotated web-based database of preimplantation gene expression with an in-built capacity for comparison of expression profiles. Comparison of gene expression profiles obtained for each developmental stage show statistically significant changes in gene expression during preimplantation development. These in silico-generated profiles were validated using RT-PCR.
为了理解早期哺乳动物发育,有必要比较植入前小鼠胚胎不同阶段的基因表达谱。我们在此描述一种利用美国国立卫生研究院(NIH)的UniGene数据库中保存的基因表达数据的方法。完整的小鼠UniGene数据库(版本号151)包含从约410万个序列生成的43104个基因簇。用于构建UniGene的表达序列标签(EST)来自cDNA文库,这些文库分别保存在表达序列标签数据库(dbEST)中,并拥有自己的目录编号。小鼠dbEST数据库包含32个从植入前阶段(未受精卵母细胞、受精卵母细胞、2细胞、4细胞、8细胞、16细胞胚胎和囊胚)构建的非标准化dbEST文库。这些文库包含映射到15731个UniGene簇的219852个EST序列。我们开发了一种计算流程方法,用于导入和汇总这些dbEST文库中包含的基因表达清单。它利用这些数据构建一个基于网络的植入前基因表达注释数据库,并具有比较表达谱的内置功能。对每个发育阶段获得的基因表达谱进行比较,结果显示在植入前发育过程中基因表达有统计学上的显著变化。这些通过计算机生成的谱图通过逆转录聚合酶链反应(RT-PCR)进行了验证。