*Y.K. and S.K. contributed equally to this work
Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3′-truncated enhanced GFP (HygR-5′ EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5′ EGFP were efficiently established from two human somatic cell lines. Established HygR-5′ EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5′ EGFP gene. With the HygR-5′ EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5′ EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.
Gene targeting in human somatic cells is a powerful technology allowing for the biological analyses of human gene function in physiological settings. Gene targeting is achieved by homologous recombination (HR) between the endogenous gene loci and targeting vectors introduced into cells. The use of gene-targeted human cell systems is particularly advantageous for the study of genetic changes leading to human diseases such as cancer (1, 2). These cell systems are also expected to serve as a platform for compound screening and thereby facilitate the development and validation of molecularly targeted therapies (3, 4).
Although gene targeting in human cells has improved significantly (5-9), the execution of gene targeting in human somatic cell lines continues to be labor-intensive and difficult to achieve depending on the genomic loci targeted and the cell lines used. This is probably because most somatic cells have low HR efficiency compared with embryonic stem cells as shown with murine myoblasts (10). To further improve this technology, it is critical to first have a robust and efficient research system to measure gene targeting efficiency in human somatic cell lines. In practice, the ratio of homologous to random integration (H/R ratio) of targeting vectors should be closely monitored, as a high H/R ratio is critical for efficient gene targeting.
In previous studies, monitoring of gene targeting efficiency has been performed using vectors carrying reporter genes such as enhanced GFP (EGFP), neomycin phosphotransferase (NeoR), and alkaline phosphatase genes that harbor intragenic inactivating mutations, deletions, or small insertions (5, 11, 12). Cells are first transduced with these reporter vectors and then with targeting vectors containing partial fragments of wild-type reporter genes that correct the inactivated reporter genes via HR and reconstitute intact reporter function. However, one concern with these reporter systems is unstable and insufficient expression of the reporter genes. Because reporter genes are generally driven by viral promoters, their expression tends to diminish over time probably because of epigenetic changes within the viral promoters that arise during the establishment and maintenance of cell clones (13-15).
In this study, we sought to overcome this problem by using hygromycin B phosphotransferase (HygR) fused with 3′-truncated EGFP (HygR-5′ EGFP) as a reporter gene. If the HygR-5′ EGFP gene is epigenetically silenced in a fraction of a reporter cell clone, this fraction will be eliminated by hygromycin selection. To evaluate the utility of this HygR-5′ EGFP reporter system, we assayed the H/R ratios of targeting vectors in human cell lines in several experimental settings.Materials and methods Vector construction
For the creation of the HygR-5′ EGFP reporter vector, the 5′ portion of EGFP (500 bp) was amplified by PCR and ligated to an EcoRI-BamHI site of pIRESneo3 (BD Biosciences, Franklin Lakes, NJ, USA). A BstXI-XbaI fragment containing a synthetic intron, internal ribosomal entry site (IRES), and NeoR was then deleted from the resulting plasmid, and the remaining fragment was blunt-ended and self-ligated. This plasmid was then digested with NheI and AgeI and ligated to the PCR-amplified HygR coding region with a Kozak consensus sequence (GCCACC) at its 5' terminus, so that the HygR-5' EGFP was translated in frame. Finally, the 3' homology arm consisting of the internal portion of simian virus 40 (SV40) large T (1 kb) was PCR-amplified and inserted into a BstZ17I site of the plasmid.
To construct ATG-less targeting vector (TV), pAAV-MCS (Agilent Technologies, Santa Clara, CA, USA) was digested with NotI, and the backbone containing two inverted terminal repeats was recovered. Multiple restriction enzyme sites, including the NotI and XhoI sites, were then incorporated into this backbone, destroying the original NotI sites and thus generating “modified pAAV-MCS.” Meanwhile, an EGFP coding region without an authentic start codon was PCR-amplified and inserted into an XbaI site of pSEPT (a gift from Dr. Fred Bunz) (16) in the forward direction. The resulting plasmid was cut with NotI and XhoI, and the isolated NotI-XhoI fragment containing ATG-less EGFP and a promoter-trap assembly was ligated to a NotI-–XhoI site of the modified pAAV-MCS. The resulting plasmid was finally digested with XhoI and ligated to an XhoI fragment from the HygR-5′ EGFP reporter consisting of the internal portion of SV40 large T (i.e., the 3′ homology arm of the HygR-5′ EGFP reporter).