Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
BMC Biotechnol. 2012 Jan 16;12:3. doi: 10.1186/1472-6750-12-3.
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.
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.
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.
能够进行逆转录病毒转导的克隆载体使许多有丝分裂细胞系能够稳定过表达基因。然而，如果靶 cDNA 插入物中也存在这些序列，那么它们克隆位点中相对较少的可行限制酶序列可能会阻碍过表达构建体的成功生成。
我们利用连接酶独立克隆（LIC）技术，对高效的逆转录病毒转导载体 pBABE 进行了修饰，使其不再依赖于限制酶进行克隆。相反，修饰后的质粒 pBLIC 利用 T4 DNA 聚合酶 3'外切核酸酶活性产生的随机 12/13 碱基突出端。通过将互补序列基于 PCR 引入感兴趣的任何 cDNA 中，即可将其普遍克隆到 pBLIC 中。在此，我们描述了 pBLIC 质粒的构建，并通过将 Bax、过氧化氢酶和 p53 三种不同的 cDNA 转导到人前列腺癌细胞系 LNCaP 或人肺癌细胞系 H358 中，证明了其成功的克隆和蛋白过表达。
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 插入物内部限制位点的要求。因此，它构成了一种有效的逆转录病毒克隆系统，适用于实验室规模的稳定基因过表达或高通量应用，如创建逆转录病毒 cDNA 文库。据我们所知，pBLIC 是第一个用于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。
