GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos
GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos
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DOI：
10.1002/dvdy.10503
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发表时间：
2004-03-01
影响因子：
2.5
通讯作者：
Antin, PB
中科院分区：
文献类型：
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作者：
Bell, GW;Yatskievych, TA;Antin, PB
尽管研究人员可以获得的基因组序列的质量和数量不断增加，但从序列信息预测基因功能仍然是一个挑战。一种快速了解新基因潜在功能作用的方法是通过基因表达图谱。我们进行了高通量的整体原位杂交（ISH）筛选鸡胚，以确定新的，差异表达的基因。从Hamburger和汉密尔顿（HH）4-7期（晚期原肠胚）鸡胚内胚层-中胚层文库的cDNA克隆产生约1，200个5'表达序列标签（EST）。在筛选以去除普遍表达的cDNA和内部聚类后，并在与GenBank序列比较后，通过自动化高通量ISH筛选剩余cDNA（代表表征的和未表征的基因）在HH 3-14期胚胎中的表达。在成功进行ISH的786个cDNA中，约30%显示普遍表达，40%为阴性，约30%显示限制性表达模式。鉴定了在每个胚胎区域（包括原条、体节、发育中的心血管系统和神经管/神经嵴）中显示限制性表达的cDNA。开发了一个关系数据库，以保存所有EST序列、ISH图像和相应的BLAST报告信息，并使数据的浏览和查询成为可能。用户界面可以在http://geisha.biosci.arizona.edu上免费访问。结果表明，高通量的整体包埋ISH提供了一种有效的方法来确定新的基因，差异表达的发育中的鸡胚胎。发展动力学229.677-687，2004年。(C)2004 Wiley-Liss，Inc.
Despite the increasing quality and quantity of genomic sequence that is available to researchers, predicting gene function from sequence information remains a challenge. One method for obtaining rapid insight into potential functional roles of novel genes is through gene expression mapping. We have performed a high throughput whole-mount in situ hybridization (ISH) screen with chick embryos to identify novel, differentially expressed genes. Approximately 1,200 5' expressed sequence tags (ESTs) were generated from cDNA clones of a Hamburger and Hamilton (HH) stage 4-7 (late gastrula) chick embryo endoderm-mesoderm library. After screening to remove ubiquitously expressed cDNAs and internal clustering and after comparison to GenBank sequences, remaining cDNAs (representing both characterized and uncharacterized genes) were screened for expression in HH stage 3-14 embryos by automated high throughput ISH. Of 786 cDNAs for which ISH was successfully performed, approximately 30% showed ubiquitous expression, 40% were negative, and approximately 30% showed a restricted expression pattern. cDNAs were identified that showed restricted expression in every embryonic region, including the primitive streak, somites, developing cardiovascular system and neural tube/neural crest. A relational database was developed to hold all EST sequences, ISH images, and corresponding BLAST report information, and to enable browsing and querying of data. A user interface is freely accessible at http://geisha.biosci.arizona.edu. Results show that high throughput whole-mount ISH provides an effective approach for identifying novel genes that are differentially expressed in the developing chicken embryo. Developmental Dynamics 229.677-687, 2004. (C) 2004 Wiley-Liss, Inc.