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首页> 外文期刊>BMC Biotechnology >Creation and validation of a ligation-independent cloning (LIC) retroviral vector for stable gene transduction in mammalian cells
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Creation and validation of a ligation-independent cloning (LIC) retroviral vector for stable gene transduction in mammalian cells

机译：用于哺乳动物细胞中稳定基因转导的非连接依赖性克隆（LIC）逆转录病毒载体的创建和验证

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Background Cloning vectors capable of retroviral transduction have enabled stable gene overexpression in numerous mitotic cell lines. However, the relatively small number of feasible restriction enzyme sequences in their cloning sites can hinder successful generation of overexpression constructs if these sequences are also present in the target cDNA insert. Results Utilizing ligation-independent cloning (LIC) technology, we have modified the highly efficient retroviral transduction vector, pBABE, to eliminate reliance on restriction enzymes for cloning. Instead, the modified plasmid, pBLIC, utilizes random 12/13-base overhangs generated by T4 DNA polymerase 3' exonuclease activity. PCR-based introduction of the complementary sequence into any cDNA of interest enables universal cloning into pBLIC. Here we describe creation of the pBLIC plasmid, and demonstrate successful cloning and protein overexpression from three different cDNAs, Bax , catalase, and p53 through transduction into the human prostate cancer cell line, LNCaP or the human lung cancer line, H358. Conclusions Our results show that pBLIC vector retains the high transduction efficiency of the original pBABE while eliminating the requirement for checking individual cDNA inserts for internal restriction sites. Thus it comprises an effective retroviral cloning system for laboratory-scale stable gene overexpression or for high-throughput applications such as creation of retroviral cDNA libraries. To our knowledge, pBLIC is the first LIC vector for retroviral transduction-mediated stable gene expression in mammalian cells.
机译：能够逆转录病毒转导的背景克隆载体已使许多有丝分裂细胞系中的基因稳定地过表达。但是，如果在目标cDNA插入片段中也存在这些序列，那么在它们的克隆位点中相对较少的可行限制酶序列可能会阻碍成功生成过表达构建体。结果利用连接非依赖性克隆（LIC）技术，我们修饰了高效逆转录病毒转导载体pBABE，以消除对限制酶的依赖。相反，修饰的质粒pBLIC利用T4 DNA聚合酶3'核酸外切酶活性产生的随机12/13碱基突出端。基于PCR的互补序列导入任何目标cDNA均可将其克隆到pBLIC中。在这里，我们描述了pBLIC质粒的创建，并证明了通过转导进入人前列腺癌细胞系LNCaP或人肺癌细胞系H358，成功地从三种不同的cDNA Bax，过氧化氢酶和p53进行克隆和蛋白质过表达。结论我们的结果表明，pBLIC载体保留了原始pBABE的高转导效率，同时消除了检查单个cDNA插入片段内部限制性位点的需求。因此，它包括一个有效的逆转录病毒克隆系统，可用于实验室规模的稳定基因过表达或用于高通量应用，例如创建逆转录病毒cDNA文库。据我们所知，pBLIC是第一个在哺乳动物细胞中逆转录病毒转导介导的稳定基因表达的LIC载体。

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