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首页> 外文期刊>Gene Therapy and Molecular Biology >Ribozyme-dependent inactivation of lacZ mRNA in E. coli: a feasibility study to set up a rapid in vivo systemfor screening HIV-1 RNA-specific ribozymes
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Ribozyme-dependent inactivation of lacZ mRNA in E. coli: a feasibility study to set up a rapid in vivo systemfor screening HIV-1 RNA-specific ribozymes

机译：大肠杆菌中lacZ mRNA的核酶依赖性灭活：建立快速体内系统以筛选HIV-1 RNA特异性核酶的可行性研究

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Ribozymes are potentially useful tools with widespread applications in gene therapy of several diseases. In order to assess the in vivo cleavage efficiency of human immunodeficiency virus (HIV)-1 RNA-specific ribozymes, a bacterial indicator cell system could be developed in which the degree of inhibition of β-galactosidase activity would correlate with ribozyme activity. The suitability of this indicator cell system was assessed using a ribozyme targeted against the env coding region within the HIV-1 RNA. To this end, a pGEM4Z-based plasmid was engineered wherein oligodeoxynucleotides containing a hammerhead ribozyme and its target site were cloned in frame within the lacZ coding region that encodes for the α fragment of β-galactosidase. Extra nucleotides were included in the insert to ensure that the lacZ open reading frame was not interrupted due to a frameshift or nonsense mutation. In E. coli indicator cells harbouring this plasmid, ribozyme-mediated cleavage of the target site provided in cis and the subsequent loss of β-galactosidase activity should correlate with ribozyme activity. However, frameshift mutations were observed upon sequence analysis of plasmid DNA isolated from the selected light blue to white colonies. Because these mutations affected the production of the β-galactosidase α fragment, a direct correlation between β-galactosidase and ribozyme activities could not be established in vivo. Thus, in clones which demonstrated visibly lower β-galactosidase activities than the control, the effect of the frameshift mutations on lacZ mRNA translation can not be discounted. In clones expressing ribozymes but displaying dark blue colour, it is possible that lacZ mRNAs were cleaved but that the β-galactosidase substrates used were sensitive enough to allow detection of proteins translated from residual lacZ mRNA transcripts. The use of alternative β-galactosidase substrates with less sensitivity may enable the use of the proposed indicator cell system.
机译：核酶是在多种疾病的基因治疗中广泛应用的潜在有用工具。为了评估人类免疫缺陷病毒（HIV）-1 RNA特异性核酶的体内裂解效率，可以开发一种细菌指示剂细胞系统，其中β-半乳糖苷酶活性的抑制程度与核酶活性相关。使用针对HIV-1 RNA中env编码区的核酶评估了该指示剂细胞系统的适用性。为此，设计了基于pGEM4Z的质粒，其中将含有锤头状核酶的寡聚脱氧核苷酸及其靶位点按框克隆在编码β-半乳糖苷酶α片段的lacZ编码区内。插入片段中包含额外的核苷酸，以确保lacZ开放阅读框不会因移码或无义突变而中断。在带有该质粒的大肠杆菌指示剂细胞中，核酶介导的顺式靶位点的切割以及随后β-半乳糖苷酶活性的丧失应与核酶活性相关。然而，在对从选定的浅蓝色至白色菌落分离的质粒DNA进行序列分析时观察到移码突变。因为这些突变影响β-半乳糖苷酶α片段的产生，所以不能在体内建立β-半乳糖苷酶和核酶活性之间的直接关联。因此，在表现出明显比对照低的β-半乳糖苷酶活性的克隆中，移码突变对lacZ mRNA翻译的影响不可小视。在表达核酶但显示深蓝色的克隆中，lacZ mRNA可能被切割，但所用的β-半乳糖苷酶底物足够灵敏，足以检测从残留lacZ mRNA转录本翻译的蛋白质。使用敏感性较低的替代性β-半乳糖苷酶底物可以使所建议的指示剂细胞系统得以使用。

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