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首页> 外文期刊>Gene Therapy and Molecular Biology >A novel system for selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli
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A novel system for selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli

机译：一种使用二氢叶酸还原酶（DHFR）基因作为大肠杆菌中选择标记的细胞内活性核酶选择系统

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If ribozymes are to be exploited in vivo, it is necessary to select ribozymes that are functional in the intracellular environment. Ribozymes selected in the intracellular environment should retain their function in vivo as well as in vitro. We have devised a novel system for selection of active ribozymes from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In the DHFR expression vector, a sequence encoding either an active or an inactive ribozyme was connected either upstream (5'-connected active or inactive ribozyme) or downstream (3'-connected active or inactive ribozyme) of the gene for DHFR. Each plasmid was designed such that, when the ribozyme was active, the ribozyme would cleave the target site and, as a result, the rate of production of DHFR would be high enough to endow resistance to trimethoprim (TMP). In the case of both 5'-connected and 3'-connected ribozymes, the active ribozyme did indeed cleave the primary transcript in vivo, whereas inactive ribozymes had no cleavage activity. We confirmed that cells that harbored the active ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored an inactive ribozyme-coding plasmid. Consequently, when cells were transformed with a mixture that consisted of active ribozyme-coding and inactive ribozyme-coding plasmids at a ratio of 1:1, it was mainly the cells that harbored the active ribozyme that survived in the presence of TMP. These results indicated that our positive selection system in vivo was functional and that, moreover, if the background "noise" could be removed completely in the future, it might usefully complement existing selection systems in vitro.
机译：如果要在体内利用核酶，则必须选择在细胞内环境中起作用的核酶。在细胞内环境中选择的核酶应在体内和体外保持其功能。我们设计了一种新颖的系统，使用二氢叶酸还原酶（DHFR）基因作为选择标记，从活性和非活性核酶库中选择活性核酶。在DHFR表达载体中，将编码活性或无活性核酶的序列连接到DHFR基因的上游（5'-连接的活性或无活性核酶）或下游（3'-连接的活性或无活性核酶）。设计每个质粒，使得当核酶具有活性时，核酶将切割靶位点，结果，DHFR的产生速率将足够高以赋予对甲氧苄啶（TMP）的抗性。对于5'连接的核酶和3'连接的核酶，活性核酶确实确实在体内切割了初级转录物，而无活性的核酶则没有切割活性。我们证实，在固定浓度的TMP存在下，具有活性核酶编码质粒的细胞比具有无活性核酶编码质粒的相应细胞生长更快。因此，当用由活性核酶编码质粒和无活性核酶编码质粒以1：1的比例组成的混合物转化细胞时，在TMP存在下，主要保留有活性核酶的细胞得以存活。这些结果表明我们的体内阳性选择系统是有功能的，此外，如果将来可以完全消除背景“噪声”，它可能会有用地补充现有的体外选择系统。

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