Abstract
BackgroundGene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO) vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process.ResultsWe have developed a one-step cloning method for the insertion of two arms into a KO vector using exonuclease III. We modified an adeno-associated virus KO shuttle vector (pTK-LoxP-NEO-AAV) to yield pAAV-LIC, which contained two cassettes at the two multiple-cloning sites. The vector was digested with EcoRV to give two fragments. The two homologous arms, which had an overlap of 16 bases with the ends of the vector fragments, were amplified by polymerase chain reaction. After purification, the four fragments were mixed and treated with exonuclease III, then transformed into Escherichia coli to obtain the desired clones. Using this method, we constructed SirT1 and HDAC2 KO vectors, which were used to establish SirT1 KO cells from the colorectal cancer cell line (HCT116) and HDAC2 KO cells from the colorectal cancer cell line (DLD1).ConclusionsOur method is a fast, simple, and efficient technique for cloning, and has great potential for high-throughput construction of KO vectors.
Highlights
Gene targeting is a powerful method that can be used for examining the functions of genes
To increase cloning efficiency and ligation of four fragments simultaneously, we developed a one-step ligation-independent cloning (LIC) method for construction of KO vectors
Compared with the traditional ‘digestion-ligation’ vector construction method, our approach is not limited by the availability of restriction sites; somatic cell gene KO vectors can be constructed within 2–3 days
Summary
Gene targeting is a powerful method that can be used for examining the functions of genes. The construction of knockout (KO) vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Gene targeting is a powerful method for the production of genetically modified cell lines or animals, allowing for the various functions of genes to be studied. Several thousand genes have been disrupted using homologous recombination. When these methods have been applied to human somatic cells they have generally been ineffective because of very low targeting efficiencies [1]. We believe it is essential to improve the efficiency of knockout (KO) approaches in human somatic cells. Hirata [2] and Porteus [3]
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