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首页> 外文期刊>Developmental biology >Developmental effector gene regulation: Multiplexed strategies for functional analysis
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Developmental effector gene regulation: Multiplexed strategies for functional analysis

机译：发育效应基因调控：功能分析的多路复用策略

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The staggering complexity of the genome controls for developmental processes is revealed through massively parallel cis-regulatory analysis using new methods of perturbation and readout. The choice of combinations of these new methods is tailored to the system, question and resources at hand. Our focus is on issues that include the necessity or sufficiency of given cis-regulatory modules, cis-regulatory function in the normal spatial genomic context, and easily accessible high throughput and multiplexed analysis methods. In the sea urchin embryonic model, recombineered BACs offer new opportunities for consecutive modes of cis-regulatory analyses that answer these requirements, as we here demonstrate on a diverse suite of previously unstudied sea urchin effector genes expressed in skeletogenic cells. Positively active cis-regulatory modules were located in single Nanostring experiments per BAC containing the gene of interest, by application of our previously reported “barcode” tag vectors of which?100 can be analyzed at one time. Computational analysis of DNA sequences that drive expression, based on the known skeletogenic regulatory state, then permitted effective identification of functional target site clusters. Deletion of these sub-regions from the parent BACs revealed module necessity, as simultaneous tests of the same regions in short constructs revealed sufficiency. Predicted functional inputs were then confirmed by site mutations, all generated and tested in multiplex formats. There emerged the simple conclusion that each effector gene utilizes a small subset of inputs from the skeletogenic GRN. These inputs may function to only adjust expression levels or in some cases necessary for expression. Since we know the GRN architecture upstream of the effector genes, we could then conceptually isolate and compare the wiring of the effector gene driver sub-circuits and identify the inputs whose removal abolish expression.
机译：通过使用新的扰动和读数的新方法，通过大规模平行的顺式调节分析揭示了发展过程基因组对照的惊人复杂性。这些新方法的组合的选择是针对系统，问题和资源量身定制的。我们的重点是在正常空间基因组背景下包括鉴定顺式规范模块，CIS-Convisty功能的必要性或充足的问题，易于访问的高通量和多路复用分析方法。在海胆胚胎模型中，重组BAC为连续模式提供新的机会，即支持这些要求的连续式分析，正如我们在这里在骨科细胞中表达的先前未捕获的海胆效应基因多样化的套件上。正极性的顺式调节模块位于每种BAC的单一纳米型实验中，含有目的的基因，通过我们先前报道的“条形码”标签载体，其中可以一次分析> 100。基于已知的骨膜原调节状态，驱动表达的DNA序列的计算分析，然后允许有效鉴定功能靶位簇。从父母BAC删除这些子区域揭示了模块必要性，因为在短的构建中的同一区域的同时测试显示充足。然后通过站点突变确认预测的功能输入，全部以多路复用格式生成和测试。出现了每个效应基因的简单结论，即每个效应基因利用来自骨膜原子的小的输入子集。这些输入可以仅用于调整表达水平或在表达所需的某些情况下。由于我们知道效应基因上游的GRN结构，因此我们可以在概念上隔离并比较效应器基因驱动器子电路的布线并识别除去废除表达的输入。

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