Microarray transfection analysis of transcriptional regulation by cAMP-dependent protein kinase
Microarray transfection analysis of transcriptional regulation by cAMP-dependent protein kinase
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DOI：
10.1074/mcp.m400018-mcp200
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发表时间：
2004-08-01
影响因子：
7
通讯作者：
Uhler, MD
中科院分区：
文献类型：
--
作者：
Redmond, TM;Ren, XM;Uhler, MD
基于基因组序列分析和微阵列杂交研究，已经描述了多种生物信息学工具来表征潜在的转录调控机制。然而，这些调控机制仍在实验中使用瞬时转染方法进行验证。目前的转染方法受限于它们的大规模和对某些细胞类型的低效率水平。我们的目标是开发一种基于微阵列的转染方法，该方法可以针对不同的细胞类型进行优化，并可用于转录调节的报告基因分析。在这里，我们描述了一种新的转染方法，称为STEP（表面转染和表达协议），它采用基于微阵列的贴壁细胞DNA转染来分析转录调控的功能。在STEP中，将具有生物活性的重组蛋白与表达载体dna复合，然后在显微镜载玻片上定位。STEP复合体中使用的重组蛋白可以改变，以提高不同细胞类型的效率。我们证明STEP有效地转染超螺旋质粒和pcr生成的线性表达盒。使用编码camp依赖性蛋白激酶（PKA）的效应表达载体以及含有PKA调控启动子的报告载体进行的共转染实验表明，STEP转染可以检测和定量该蛋白激酶的转录调控。由于生物信息学研究经常导致许多假定的调控元件和信号通路的鉴定，这种方法应该在转录调控的高通量功能基因组研究中实用。
A wide variety of bioinformatic tools have been described to characterize potential transcriptional regulatory mechanisms based on genomic sequence analysis and microarray hybridization studies. However, these regulatory mechanisms are still experimentally verified using transient transfection methods. Current transfection methods are limited both by their large scale and by the low level of efficiency for certain cell types. Our goals were to develop a microarray-based transfection method that could be optimized for different cell types and that would be useful in reporter assays of transcriptional regulation. Here we describe a novel transfection method, termed STEP (surface transfection and expression protocol), which employs microarray-based DNA transfection of adherent cells in the functional analysis of transcriptional regulation. In STEP, recombinant proteins with biological activities designed to enhance transfection are complexed with expression vector DNAs prior to spotting on microscope slides. The recombinant proteins used in STEP complexes can be varied to increase the efficiency for different cell types. We demonstrate that STEP efficiently transfects both supercoiled plasmids and PCR-generated linear expression cassettes. A co-transfection assay using effector expression vectors encoding the cAMP-dependent protein kinase (PKA), as well as reporter vectors containing PKA-regulated promoters, showed that STEP transfection allows detection and quantitation of transcriptional regulation by this protein kinase. Because bioinformatic studies often result in the identification of many putative regulatory elements and signaling pathways, this approach should be of utility in high-throughput functional genomic studies of transcriptional regulation.