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BMC Biotechnol. 2012 Jan 16;12:3. doi: 10.1186/1472-6750-12-3.

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

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1
Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

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.

PMID:
22248071
PMCID:
PMC3298557
DOI:
10.1186/1472-6750-12-3
[Indexed for MEDLINE]
Free PMC Article
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