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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Gene targeting using a promoterless gene trap vector ('targeted trapping') is an efficient method to mutate a large fraction of genes
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Gene targeting using a promoterless gene trap vector ('targeted trapping') is an efficient method to mutate a large fraction of genes

机译：使用无启动子的基因捕获载体进行基因靶向（“靶向捕获”）是使大部分基因突变的有效方法

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摘要

A powerful tool for postgenomic analysis of mammalian gene function is gene targeting in mouse ES cells. We report that homologous recombination using a promoterless gene trap vector ("targeting trapping") yields targeting frequencies averaging above 50%, a significant increase compared with current approaches. These high frequencies appear to be due to the stringency of selection with promoterless constructs, because most random insertions are silent and eliminated by drug selection. The promoterless design requires that the targeted gene be expressed in ES cells at levels exceeding a certain threshold (which we estimate to be approximate to 1% of the transferrin receptor gene expression level, for the secretory trap vector used here). Analysis of 127 genes that had been trapped by random (nontargeted) gene trapping with the same vector shows that virtually all are expressed in ES cells above this threshold, suggesting that targeted and random trapping share similar requirements for expression levels. In a random sampling of 130 genes encoding secretory proteins, about half were expressed above threshold, suggesting that about half of all secretory genes are accessible by either targeted or random gene trapping. The simplicity and high efficiency of the method facilitate systematic targeting of a large fraction of the genome by individual investigators and large-scale consortia alike.
机译：哺乳动物基因功能的后基因组分析的强大工具是小鼠ES细胞中的基因靶向。我们报道了使用无启动子的基因陷阱载体（“目标诱捕”）进行的同源重组产生的目标频率平均超过50％，与当前方法相比显着增加。这些高频率似乎是由于无启动子构建体选择的严格性所致，因为大多数随机插入都是沉默的，并且通过药物选择得以消除。无启动子的设计要求靶向基因在ES细胞中的表达水平超过某个阈值（对于此处使用的分泌性捕获载体，我们估计其约为转铁蛋白受体基因表达水平的1％）。对使用相同载体进行的随机（非靶向）基因捕获所捕获的127个基因的分析表明，实际上所有基因均在高于该阈值的ES细胞中表达，这表明靶向和随机捕获对表达水平具有相似的要求。在130个编码分泌蛋白的基因的随机抽样中，大约一半的表达高于阈值，这表明所有分泌基因中的大约一半可通过靶向或随机基因捕获来访问。该方法的简单性和高效性促进了单个研究者和大规模联合体对大部分基因组的系统靶向。

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