Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning.
Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning.
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DOI：
10.2144/000114265
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发表时间：
2015-03
期刊：
影响因子：
2.7
通讯作者：
Ott DE
中科院分区：
文献类型：
--
作者：
Coren LV;Jain S;Trivett MT;Ohlen C;Ott DE
在这里，我们提出了一种改进的策略，利用“金门”克隆策略生产表达t细胞受体（TCR）的逆转录病毒载体。该方法利用了TCR基因的模块化特性，直接从RNA或cDNA中扩增出TCR α和β可变区，然后将其与各自的恒定区基因克隆融合到逆转录病毒TCR表达载体中。与传统的三步过程相比，这种一步的方法大大简化了TCR载体的生产过程，传统的三步过程通常包括克隆整个TCR基因，生产TCR表达盒，构建逆转录病毒结构。到目前为止，我们已经制作了TCR载体，将7个功能性的人/恒河猴TCR转移到原代T细胞中用于该方法的研究。这种方法有望将其他基因与确定的可变区域（如免疫球蛋白）组装在一起。
Here, we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a “Golden Gate” cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. This one step approach greatly streamlines the TCR vector production process versus the traditional three-step process that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have produced TCR vectors which transferred 7 functional human/rhesus macaque TCRs into primary T cells for studies using this method. This approach has promise to assemble other genes with defined variable regions such as immunoglobulins.