Tanaka T S, Jaradat S A, Lim M K, Kargul G J, Wang X, Grahovac M J, Pantano S, Sano Y, Piao Y, Nagaraja R, Doi H, Wood W H, Becker K G, Ko M S
Laboratory of Genetics and DNA Array Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-6820, USA.
Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):9127-32. doi: 10.1073/pnas.97.16.9127.
cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.
cDNA微阵列技术已越来越多地用于监测各种组织和细胞类型中的全局基因表达模式。然而,由于缺乏合适的cDNA文库,尤其是针对早期发育阶段的文库,该技术在哺乳动物发育研究中的应用受到了阻碍。为克服这一问题,采用了一种基于PCR的cDNA文库构建方法,从植入前和植入周边胚胎、胚胎第12.5天的雌性性腺/中肾以及新生卵巢中获得了52374个表达序列标签。从这些cDNA文库中,组装了一个代表15264个独特基因(78%为新基因,22%为已知基因)的微阵列。在最初的应用中,评估了胎盘和胚胎基因表达谱的差异。在发育的第12.5天阶段,基于三次重复实验,720个基因(6.5%)在胎盘和胚胎之间的表达显示出统计学上的显著差异。在胎盘中表达更高的289个基因中,61个胎盘特异性基因编码了例如一种新型催乳素样蛋白。由此,胎盘高表达(且通常是特异性)基因的数量比之前报道的总数增加了5倍,这说明了微阵列在组织特异性基因发现和哺乳动物发育程序分析方面的潜力。