Retroviral vectors to monitor somatic hypermutation
Retroviral vectors to monitor somatic hypermutation
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DOI：
10.1016/j.jim.2005.02.015
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发表时间：
2005-05-01
影响因子：
2.2
通讯作者：
Wabl, M
中科院分区：
文献类型：
--
作者：
Klasen, M;Spillmann, FJX;Wabl, M
最近对超突变研究的扩展可能受益于一种快速而简单的测量突变率的方法。在本文中，我们比较了不同的逆转录病毒载体设计监测体内超突变。逆转录病毒载体联合收割机将高转导率与在宿主细胞基因组内随机位点的整合相结合，从而平衡对报道基因的位置效应。该载体含有一个报告基因与提前TAG终止密码子;在回复，全长荧光蛋白的表达。琥珀密码子上的任何一个点突变都会激活终止密码子--除了从G到A的转变，它只产生终止密码子TAA。在构建体中，报告基因之后是内部核糖体进入位点和允许选择稳定转导的细胞的第二标记。作为报告基因，我们测试了绿色和黄色荧光蛋白（GFP和YFP）;以及具有红色荧光的各种蛋白（dsRed）。第二个标记是抗药性基因或第二种荧光蛋白。我们还将各种顺式作用增强子元件引入报告构建体中，以研究增强子对转录和超突变的同时活性。我们发现，GFP作为报告基因，结合药物选择标记，给出了最一致和最方便的突变率测量。DsRed是GFP的良好替代物，但是当与绿色荧光测量组合时，需要具有更大荧光强度的变体。我们还证实，靶向超突变不需要免疫球蛋白特异性序列。根据它们在这些异位表达的构建体中的位置，增强子可以对超突变具有积极或消极的影响。(c)2005 Elsevier B. V.保留所有权利。
The recent expansion of studies on hypermutation may benefit from a fast and uncomplicated way to measure mutation rates. In this paper we compare different retroviral vector designs for monitoring hypermutation in vivo. Retroviral vectors combine a high transduction rate with integration at random sites within the host cell genome, thus equalizing positional effects on the reporter gene. The vectors contain a reporter gene with a premature TAG termination codon; upon reversion, a full-length fluorescent protein is expressed. Any single point mutation at the amber codon activates the reporter-except the transition from G to A, which only creates the stop codon TAA. In the construct, the reporter gene is followed by an internal ribosome entry site and a second marker that allows selection of stably transduced cells. As a reporter gene, we tested the green and yellow fluorescence proteins (GFP and YFP); and various proteins with red fluorescence (dsRed). The second marker was either a drug resistance gene, or a second fluorescent protein. We also introduced various cis-acting enhancer elements into the reporter construct, to study the simultaneous activity of enhancers on transcription and hypermutation. We found that GFP as a reporter, combined with a drug selection marker, gave the most consistent and convenient mutation rate measurements. DsRed is a good alternative to GFP, but variants with greater fluorescence intensity are needed when combined with green fluorescence measurements. We also confirm that no immunoglobulin specific sequence is needed to target hypermutation. Depending on their position in these ectopically expressed constructs, enhancers can have positive or negative effects on hypermutation. (c) 2005 Elsevier B.V. All rights reserved.