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. In mice, several thousand genes have been disrupted using homologous recombination. However, when these methods have been applied to human somatic cells they have generally been ineffective because of very low targeting efficiencies
. Although RNA interference can reduce the expression of a gene, interpretation of such experiments can be unreliable because of non-specific targeting or incomplete inactivation of genes. Therefore, we believe it is essential to improve the efficiency of knockout (KO) approaches in human somatic cells.
Recombinant adeno-associated viruses (rAAVs), such as human parvovirus, possess a single-stranded DNA genome of around 4.7 kb. Hirata
 and Porteus
 found that rAAVs can be used to target genes in human cell lines. Use of these rAAVs resulted in higher targeting frequencies than those obtained with conventional plasmid vectors
[4, 5]. The wild-type human parvovirus genome contains two open reading frames (ORFs), designated rep and cap, flanked by two inverted terminal repeats. The rep ORF encodes proteins involved in viral replication, and the cap ORF encodes proteins necessary for viral packaging. In rAAV-mediated KO vectors these ORFs are deleted and replaced with a neomycin resistant gene flanked by two homology arms. The stages involved in carrying out rAAV-mediated gene KO include: (i) design and construction of the AAV KO vector; (ii) collecting an infectious rAAV stock; (iii) infecting the appropriate cell line; (iv) screening for homologous recombinants; and (v) iteratively targeting the multiple alleles. It takes at least three months just to target the first allele
Apart from the low efficiency of homologous recombination, a rate-limiting step in gene targeting of human somatic cell lines is assembly of the gene targeting construct. Traditionally, this requires an amplification step to obtain two large homologous fragments of genomic DNA, followed by restriction endonuclease digestion, and then numerous cloning steps. It is an extremely time-consuming process and limited by the available unique restriction enzyme sites in the vector and in the two amplified homologous fragments. Phage-based Escherichia coli homologous recombination systems
[7–9] have been developed that now make it possible to subclone or modify DNA cloned into plasmids, bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs) without the need for restriction enzymes or DNA ligases. However, these recombination systems require long homology arms and usually one can only insert one fragment at the time.
Traditional DNA cloning suffers from several limitations, including poor ligation efficiency, along with a dependence on the availability of unique restriction sites in both the insert and vector. For this reason, many methods regarding directional subcloning have been developed
[10, 11]. These methods include the use of uracil DNA N-glycosylase
, T4 DNA polymerase
, enzymatic assembly
[14–16] or exonuclease III (ExoIII)
 to generate long compatible cohesive ends between the DNA insert and cloning vector. It has also been found that by generating longer cohesive ends, the annealed DNA complex becomes more stable and the ligation reaction can be omitted. This technique has come to be known as ligation-independent cloning (LIC), and has been demonstrated to have a high efficiency. Developing more LIC methods will give the researchers more chioce according to the different situations.
To increase cloning efficiency and ligation of four fragments simultaneously, we developed a one-step LIC method for construction of KO vectors. Using this method, it becomes easy to construct KO vectors in two days. In this paper, we have outlined our strategy for modifying pTK-LoxP-NEO-AAV, and demonstrated successful construction of a SirT1 and HDAC2 KO vector. Once we had made the vectors, we were able to obtain SirT1 KO cells from the colorectal cancer cell line HCT116 and HDAC2 KO cells from the colorectal cancer cell line DLD1.