Identification of the genomic insertion site of Pmel-1 TCR α and β transgenes by next-generation sequencing.
Identification of the genomic insertion site of Pmel-1 TCR α and β transgenes by next-generation sequencing.
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DOI：
10.1371/journal.pone.0096650
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Restifo NP
中科院分区：
文献类型：
--
作者：
Ji Y;Abrams N;Zhu W;Salinas E;Yu Z;Palmer DC;Jailwala P;Franco Z;Roychoudhuri R;Stahlberg E;Gattinoni L;Restifo NP
pmel-1 T细胞受体转基因小鼠是研究肿瘤免疫、CD 8 + T细胞分化、自身免疫和过继免疫治疗机制的理想模型。转基因的“接合性”影响转基因表达水平，并可能损害最佳育种方案设计。然而，pmel-1小鼠的整合位点仍未被表征。这也适用于在快速和廉价的下一代测序的现代时代之前创建的许多其他常用的转基因小鼠。在这里，我们表明，全基因组测序可用于确定确切的pmel-1基因组整合位点，即使是相对“浅”（8X）的覆盖。结果被用来开发一个有效的聚合酶链反应为基础的基因分型试验。我们首次提供了一种快速方便的聚合酶链反应方法来确定pmel-1转基因小鼠的剂量。我们还表明，下一代测序提供了一个可行的方法来映射外来的DNA整合位点，即使当原始载体序列的信息只有部分已知。
The pmel-1 T cell receptor transgenic mouse has been extensively employed as an ideal model system to study the mechanisms of tumor immunology, CD8+ T cell differentiation, autoimmunity and adoptive immunotherapy. The ‘zygosity’ of the transgene affects the transgene expression levels and may compromise optimal breeding scheme design. However, the integration sites for the pmel-1 mouse have remained uncharacterized. This is also true for many other commonly used transgenic mice created before the modern era of rapid and inexpensive next-generation sequencing. Here, we show that whole genome sequencing can be used to determine the exact pmel-1 genomic integration site, even with relatively ‘shallow’ (8X) coverage. The results were used to develop a validated polymerase chain reaction-based genotyping assay. For the first time, we provide a quick and convenient polymerase chain reaction method to determine the dosage of pmel-1 transgene for this freely and publically available mouse resource. We also demonstrate that next-generation sequencing provides a feasible approach for mapping foreign DNA integration sites, even when information of the original vector sequences is only partially known.
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影响因子：
6
作者：
Balatoni, Cynthia E.;Dawson, David W.;Teitell, Michael A.
通讯作者：
Teitell, Michael A.
通过淋巴结消除来清除稳态细胞因子下沉，增强了采用转移的肿瘤特异性CD8+ T细胞的功效。
影响因子：
15.3
作者：
Gattinoni, Luca;Finkelstein, Steven E;Klebanoff, Christopher A;Antony, Paul A;Palmer, Douglas C;Spiess, Paul J;Hwang, Leroy N;Yu, Zhiya;Wrzesinski, Claudia;Heimann, David M;Surh, Charles D;Rosenberg, Steven A;Restifo, Nicholas P
通讯作者：
Restifo, Nicholas P
DOI：
10.1038/nrc3322
发表时间：
2012-10
期刊：
Nature reviews. Cancer
影响因子：
--
作者：
Gattinoni L;Klebanoff CA;Restifo NP
通讯作者：
Restifo NP
影响因子：
56.9
作者：
COOLEY, L;KELLEY, R;SPRADLING, A
通讯作者：
SPRADLING, A
影响因子：
2.5
作者：
Chandler, Kelly J.;Chandler, Ronald L.;Mortlock, Douglas P.
通讯作者：
Mortlock, Douglas P.