In this regard, the Moloney murine leukemia virus (MMLV)-based pBABE vector system has been widely utilized for highly efficient stable gene overexpression in a variety of different mammalian cells with negligible off-target cellular effects. To our knowledge, pBLIC is the first LIC vector for retroviral transduction-mediated stable gene expression in mammalian cells.Ĭloning vectors capable of being packaged into retroviral particles for transduction into mammalian cells provide efficient tools for stably altering the genome of dividing cells. 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. 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. 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. PCR-based introduction of the complementary sequence into any cDNA of interest enables universal cloning into pBLIC. Instead, the modified plasmid, pBLIC, utilizes random 12/13-base overhangs generated by T4 DNA polymerase 3' exonuclease activity. Utilizing ligation-independent cloning (LIC) technology, we have modified the highly efficient retroviral transduction vector, pBABE, to eliminate reliance on restriction enzymes for cloning. 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. Cloning vectors capable of retroviral transduction have enabled stable gene overexpression in numerous mitotic cell lines.
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