Creation and validation of a ligation-independent cloning (LIC) retroviral vector for stable gene transduction in mammalian cells

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.Cloning vectors capable of being packaged into retroviral particles for transduction into mammalian cells provide efficient tools for stably altering the genome of dividing cells. In this regard, the Moloney murine leukemia virus (MMLV)-based pBABE vector system [1] has been widely utilized for highly efficient stable gene overexpression in a variety of different mammalian cells with negligible off-target cellular effects. The pBABE vector consists of a bacterial origin of replication, viral elements for gene packaging, transcription and processing, and a unique restriction enzyme sequence-based cloning site http://www.addgene.org/1767/ webcite. Additionally it contains an ampicillin-resistance gene for selection in bacteria, and either puromycin, hygromycin or neomyc