Laboratory of Functional Genomics of Early Embryonic Development, Université Laval, Québec, Canada.
Mol Reprod Dev. 2011 Sep;78(9):651-64. doi: 10.1002/mrd.21364. Epub 2011 Aug 2.
While most assisted reproductive technologies (ART) are considered routine for the reproduction of species of economical importance, such as the bovine, the impact of these manipulations on the developing embryo remains largely unknown. In an effort to obtain a comprehensive survey of the bovine embryo transcriptome and how it is modified by ART, resources were combined to design an embryo-specific microarray. Close to one million high-quality reads were produced from subtracted bovine embryo libraries using Roche 454 Titanium deep sequencing technology, which enabled the creation of an augmented bovine genome catalog. This catalog was enriched with bovine embryo transcripts, and included newly discovered indel type and 3'UTR variants. Using this augmented bovine genome catalog, the EmbryoGENE Bovine Microarray was designed and is composed of a total of 42,242 probes, including 21,139 known reference genes; 9,322 probes for novel transcribed regions (NTRs); 3,677 alternatively spliced exons; 3,353 3'-tiling probes; and 3,723 controls. A suite of bioinformatics tools was also developed to facilitate microrarray data analysis and database creation; it includes a quality control module, a Laboratory Information Management System (LIMS) and microarray analysis software. Results obtained during this study have already led to the identification of differentially expressed blastocyst targets, NTRs, splice variants of the indel type, and 3'UTR variants. We were able to confirm microarray results by real-time PCR, indicating that the EmbryoGENE bovine microarray has the power to detect physiologically relevant changes in gene expression.
虽然大多数辅助生殖技术（ART）被认为是经济重要物种繁殖的常规手段，例如牛，但这些操作对胚胎发育的影响在很大程度上仍然未知。为了全面了解牛胚胎转录组以及 ART 如何改变它，我们整合资源设计了一种胚胎特异性微阵列。使用 Roche 454 Titanium 深度测序技术从减去的牛胚胎文库中产生了近一百万条高质量的读数，这使得创建增强的牛基因组目录成为可能。该目录富含牛胚胎转录本，包括新发现的插入缺失类型和 3'UTR 变体。使用这个增强的牛基因组目录，设计了 EmbryoGENE 牛微阵列，总共包含 42242 个探针，包括 21139 个已知的参考基因；9322 个用于新转录区域（NTR）的探针；3677 个可变剪接外显子；3353 个 3'端平铺探针；和 3723 个对照。还开发了一套生物信息学工具来方便微阵列数据分析和数据库创建；它包括一个质量控制模块、实验室信息管理系统（LIMS）和微阵列分析软件。在这项研究中获得的结果已经导致了鉴定差异表达的囊胚靶标、NTR、插入缺失类型的剪接变体和 3'UTR 变体。我们能够通过实时 PCR 来验证微阵列结果，表明 EmbryoGENE 牛微阵列具有检测基因表达中生理相关变化的能力。
