Identifying genes preferentially expressed in undifferentiated embryonic stem cells
Identifying genes preferentially expressed in undifferentiated embryonic stem cells
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DOI：
10.1186/1471-2121-8-37
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发表时间：
2007-08-28
期刊：
影响因子：
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通讯作者：
Leder, Philip
中科院分区：
文献类型：
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作者：
Li, Xiajun;Leder, Philip
背景资料：胚胎干细胞（ES）的维持和分化的机制是不完全understood.Results：为了解决这个问题，我们已经开发了一种逆转录病毒基因陷阱载体，可以靶向未分化的ES细胞中表达的基因。该基因捕获载体携带GFP和Neo报告基因。使用G-418药物抗性来选择其中载体整合到转录活性基因座中的ES克隆。随后进行GFP FACS分析，以鉴定GFP荧光降低的ES克隆，因此，当ES细胞分化时，转录活性降低。通过北方印迹分析证实，在我们的初步筛选中，在300个ES克隆中的6个中GFP报告基因的表达降低是在ES细胞分化过程中下调的。这六个ES克隆代表四个不同的基因。在六个整合位点中，一个在Zfp-57处，已知其基因产物在未分化的ES细胞中富集。三个位于CSL/RBP-Jkappa的一种新型亚型的内含子中，该亚型编码LIN-12/Notch途径的关键转录因子。另一个位于可能编码非编码RNA转录物的基因内。最后的整合事件发生在一个位点，可能窝藏一个新的gene.Conclusion：两者合计，我们证明了使用一种新的逆转录病毒基因陷阱载体，在确定基因优先表达在未分化的ES细胞。
Background: The mechanism involved in the maintenance and differentiation of embryonic stem (ES) cells is incompletely understood.Results: To address this issue, we have developed a retroviral gene trap vector that can target genes expressed in undifferentiated ES cells. This gene trap vector harbors both GFP and Neo reporter genes. G-418 drug resistance was used to select ES clones in which the vector was integrated into transcriptionally active loci. This was then followed by GFP FACS profiling to identify ES clones with reduced GFP fluorescence and, hence, reduced transcriptional activity when ES cells differentiate. Reduced expression of the GFP reporter in six of three hundred ES clones in our pilot screening was confirmed to be down-regulated by Northern blot analysis during ES cell differentiation. These six ES clones represent four different genes. Among the six integration sites, one was at Zfp-57 whose gene product is known to be enriched in undifferentiated ES cells. Three were located in an intron of a novel isoform of CSL/RBP-Jkappa which encodes the key transcription factor of the LIN-12/Notch pathway. Another was inside a gene that may encode noncoding RNA transcripts. The last integration event occurred at a locus that may harbor a novel gene.Conclusion: Taken together, we demonstrate the use of a novel retroviral gene trap vector in identifying genes preferentially expressed in undifferentiated ES cells.