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首页> 外文期刊>Genomics >A NotI-EcoRV promoter library for studies of genetic and epigenetic alterations in mouse models of human malignancies.
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A NotI-EcoRV promoter library for studies of genetic and epigenetic alterations in mouse models of human malignancies.

机译：NotI-EcoRV启动子文库，用于研究人类恶性肿瘤小鼠模型中的遗传和表观遗传学改变。

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Aberrant promoter methylation and associated chromatin changes are primarily studied in human malignancies. Thus far, mouse models for human cancer have been rarely utilized to study the role of DNA methylation in tumor onset and progression. It would be advantageous to use mouse tumor models to a greater extent to study the role and mechanism of DNA methylation in cancer because mouse models allow manipulation of the genome, study of samples/populations with a homogeneous genetic background, the possibility of modulating gene expression in vivo, the statistical power of using large numbers of tumor samples, access to various tumor stages, and the possibility of preclinical trials. Therefore, it is likely that the mouse will emerge as an increasingly utilized model to study DNA methylation in cancer. To foster the use of mouse models, we developed an arrayed mouse NotI-EcoRV genomic library, with clones from three commonly used mouse strains (129SvIMJ, FVB/NJ, and C57BL/6J). A total of 23,040 clones representing an estimated three- to fourfold coverage of the mouse genome were arrayed in 60 x 384-well plates. We developed restriction landmark genomic scanning (RLGS) mixing gels with 32 plates to enable the cloning of methylated sequences from RLGS profiles run with NotI-EcoRV-HinfI. RLGS was used to study aberrant methylation in two mouse models that overexpressed IL-15 or c-Myc and developed either T/NK-cell leukemia or T-cell lymphomas, respectively. Careful analysis of 198 sequences showed that 188 (94.9%) identified CpG-island sequences, 132 sequences (66.7%) had homology to the 5' regions of known genes or mRNAs, and all 132 NotI-EcoRV clones were located at the same CpG islands with the predicted promoter sequences. We have also developed a modified pGL3-based luciferase vector that now contains the NotI, AscI, and EcoRV restriction sites and allows the rapid cloning of NotI-EcoRV library fragments in both orientations. Luciferase assays using NotI-EcoRV clones confirmed that the library is enriched for promoter sequences. Thus, this library will support future genetic and epigenetic studies in mouse models.
机译：主要在人类恶性肿瘤中研究异常启动子甲基化和相关的染色质变化。迄今为止，人类癌症的小鼠模型很少用于研究DNA甲基化在肿瘤发作和进展中的作用。在更大范围内使用小鼠肿瘤模型来研究DNA甲基化在癌症中的作用和机制将是有利的，因为小鼠模型允许操纵基因组，研究具有均一遗传背景的样品/种群，调节基因表达的可能性在体内，使用大量肿瘤样品，进入各种肿瘤阶段的统计能力以及临床前试验的可能性。因此，有可能该小鼠将成为研究癌症中DNA甲基化的一种越来越常用的模型。为促进小鼠模型的使用，我们开发了阵列小鼠NotI-EcoRV基因组文库，其中包含来自三种常用小鼠品系（129SvIMJ，FVB / NJ和C57BL / 6J）的克隆。将总共​​23,040个克隆（表示小鼠基因组覆盖率估计为三到四倍）排列在60 x 384孔板中。我们开发了具有32个平板的限制性地标基因组扫描（RLGS）混合凝胶，从而能够从用NotI-EcoRV-HinfI运行的RLGS谱中克隆甲基化序列。 RLGS被用于研究过表达IL-15或c-Myc并分别发展为T / NK细胞白血病或T细胞淋巴瘤的两种小鼠模型中的异常甲基化。对198个序列的仔细分析表明，有188个（94.9％）已识别的CpG岛序列，132个序列（66.7％）与已知基因或mRNA的5'区域同源，并且所有132个NotI-EcoRV克隆都位于同一CpG具有预测的启动子序列的孤岛。我们还开发了一种修饰的基于pGL3的荧光素酶载体，该载体现在包含NotI，AscI和EcoRV限制位点，并允许在两个方向上快速克隆NotI-EcoRV文库片段。使用NotI-EcoRV克隆的萤光素酶测定法证实该文库富含启动子序列。因此，该库将支持将来在小鼠模型中进行遗传和表观遗传学研究。

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