Expression profiling with arrays of randomly disrupted genes in mouse embryonic stem cells leads to in vivo functional analysis
Expression profiling with arrays of randomly disrupted genes in mouse embryonic stem cells leads to in vivo functional analysis
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DOI：
10.1073/pnas.0400604101
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发表时间：
2004-03-23
影响因子：
11.1
通讯作者：
Ishida, Y
中科院分区：
文献类型：
--
作者：
Matsuda, E;Shigeoka, T;Ishida, Y
DNA阵列能够在一次实验中分析许多基因的表达模式。然而，在DNA阵列中找到感兴趣的基因后，有时必须进行劳动密集型的基因靶向实验以进行基因功能的体内分析。另一方面，利用随机基因捕获，在小鼠胚胎干（ES）细胞中破坏和恢复数百个基因/基因候选物相对容易，但是如果仅分析核苷酸序列而不分析捕获的DNA片段的表达模式，则可能忽略潜在的重要基因破坏事件。为了联合收割机结合上述两种实验系统的优点，我们首先建立了约900个基因捕获的小鼠ES细胞克隆，然后构建了来自被破坏基因的cDNA阵列。通过使用这些阵列，我们确定了一个新的基因主要在小鼠大脑中表达，并相应的ES细胞克隆用于产生小鼠的基因的破坏等位基因的纯合子。对基因敲除小鼠的详细分析显示，基因捕获载体完全消除了其整合位点下游的基因表达。因此，通过使用这种类型的DNA阵列鉴定具有感兴趣的表达模式的基因或新的候选基因，立即允许从具有感兴趣序列的破坏的等位基因的ES细胞克隆产生敲除小鼠。
DNA arrays are capable of profiling the expression patterns of many genes in a single experiment. After finding a gene of interest in a DNA array, however, labor-intensive gene-targeting experiments sometimes must be performed for the in vivo analysis of the gene function. With random gene trapping, on the other hand, it is relatively easy to disrupt and retrieve hundreds of genes/gene candidates in mouse embryonic stem (ES) cells, but one could overlook potentially important gene-disruption events if only the nucleotide sequences and not the expression patterns of the trapped DNA segments are analyzed. To combine the benefits of the above two experimental systems, we first created approximate to900 gene-trapped mouse ES cell clones and then constructed arrays of cDNAs derived from the disrupted genes. By using these arrays, we identified a novel gene predominantly expressed in the mouse brain, and the corresponding ES cell clone was used to produced mice homozygous for the disrupted allele of the gene. Detailed analysis of the knockout mice revealed that the gene trap vector completely abolished gene expression downstream of its integration site. Therefore, identification of a gene or novel gene candidate with an interesting expression pattern by using this type of DNA array immediately allows the production of knockout mice from an ES cell clone with a disrupted allele of the sequence of interest.