title: Creation and validation of a ligation-independent cloning (LIC) retroviral vector for stable gene transduction in mammalian cells.
abstract: 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.
authors.affiliation: Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
authors.forename: Asmita
authors.initials: A
authors.lastname: Patel
authors.forename: Anisleidys
authors.initials: A
authors.lastname: Muñoz
authors.forename: Katherine
authors.initials: K
authors.lastname: Halvorsen
authors.forename: Priyamvada
authors.initials: P
authors.lastname: Rai
mesh_terms: D002374:Catalase / Q000096:biosynthesis / Q000235:genetics; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D002374:Catalase; D004262:DNA Restriction Enzymes