In this study, we developed a molecular system to monitor gene targeting efficiency in human somatic cell lines. This system uses the HygR-EGFP fusion gene for a dual purpose, namely, antibiotic selection as well as fluorescence-based monitoring of gene targeting events. When a reporter gene is expressed independent of an antibiotic resistance gene, as in conventional reporter vectors, cell clones transduced with such vectors rarely serve as potent reporter systems (Table 1). Moreover, reporter cell clones bearing such reporter vectors are prone to lose their function over time during cell culture (Figure 5). In contrast, the use of the HygR-EGFP fusion gene allows the establishment of potent reporter clones in a less labor-intensive fashion, and the resultant reporter clones retain their function over a long duration. Indeed, the HygR-5′ EGFP reporter clones were functional throughout the study period of more than 6 months.
Another strategy permitting the acquisition of potent reporter clones in an efficient fashion may be the use of IRES to join a selection marker gene and a reporter gene instead of directly fusing them (12). By maintaining reporter clones in a selective condition, this type of reporter system may also ensure robust, prolonged expression of reporter genes. However, cistrons joined by IRES do not always produce an equivalent level of proteins. It has been shown that the amounts of two proteins produced from a transcript containing IRES vary in a position-dependent fashion (20), as well as by host cell type (21, 22), presumably because translation downstream of IRES is modulated by multiple regulatory factors (23, 24). In contrast, a fusion protein in our system secures the production of exactly the same amount of antibiotic resistance and reporter proteins, thus offering more stable activity as a reporter.
Another strategy to prevent attenuation of reporter gene function is the use of an endogenous gene as a reporter. One of the endogenous genes most commonly used for this purpose is the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene on the X chromosome. In cells bearing a single HPRT1, such as diploid cells of male origin, the disruption of single HPRT1 alleles results in the loss of hypoxanthine phosphoribosyltransferase, which is selectable with an anti-metabolic nucleoside analog 6-thioguanine (6-TG) (5, 25). Furthermore, targeted correction of mutant HPRT1 can be detected by culturing cells with hypoxanthine-aminopterin-thymidine (HAT) medium (25, 26). As a drawback, however, this system cannot be utilized with cells bearing multiple X chromosomes, such as female cells and majority of aneuploid cells. In addition, cells exhibiting the microsatellite instability (MIN) phenotype are generally resistant to 6-TG (27) and are thus not eligible for HPRT1 targeting followed by 6-TG selection. In contrast, our reporter system is suitable for virtually any type of cell of male or female origin, including those with a MIN phenotype. Furthermore, our system determines gene targeting efficiency through rapid, easily performed high-throughput procedures with the aid of flow cytometry, unlike the HPRT1-based system in which gene targeting efficiency is estimated by counting colonies on tissue culture plates after incubation of the cells with and without 6-TG.
Besides determining the H/R ratio of targeting vectors, the HygR-5′ EGFP system permits estimation of the absolute gene targeting frequency. This is accomplished by counting colonies formed on tissue culture plates after antibiotic selection in parallel with flow cytometric analyses of antibiotic-resistant cells. The absolute gene targeting frequency can be estimated by multiplying the number of colonies on the plate by the GFP-positive ratio determined by flow cytometry and then dividing the resulting number by the number of cells prepared for infection of the targeting vector.
Although gene targeting in human somatic cells can be achieved by several different methodologies (12, 28-30), AAV-mediated gene targeting has an advantage in that it does not actively introduce DNA double-strand breaks, thus likely generating fewer undesired alterations in the genome. Because the technology of AAV-mediated gene targeting is simple, cost-sensitive, publicly available, and capable of modifying human somatic cell genomes with decent efficiency, it continues to be an important alternative methodology for genome editing in human cell lines. Indeed, AAV-mediated gene targeting has been utilized in many recent studies modifying genomes in human cell lines with the aim of gene functional analyses (4, 17, 31-38). As shown in this study, our HygR-5′ EGFP reporter system enables robust monitoring of the efficiency of AAV-mediated gene targeting in human somatic cells. This reporter system will thus prove helpful in improving the design of AAV-based targeting vectors and optimizing experimental conditions for gene targeting. It may also assist in screening for genes and drugs that affect the incidence of HR, potentially leading to further improvements in gene targeting technology. Such technical improvements will eventually result in the generation of highly efficient gene targeting systems readily applicable to various types of human cells.
The authors are thankful to Drs. Ben H. Park and Fred Bunz (Johns Hopkins University) for providing the reagents. This work was partly supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 22700888 to S.K., 21591225 to Y.H., and 22500999 to H.K.), Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan (S1101027 to S.K., Y.H., and H.K.), Aichi Cancer Research Foundation (to S.K., Y.H., and H.K.), and AIKEIKAI Foundation (to S.K.).
The authors declare no competing interests.
Correspondence Address correspondence to Hiroyuki Konishi, Department of Biochemistry, Aichi Medical University School of Medicine, 1-1 Yazako Karimata, Building #2, Room 362, Nagakute, Aichi, Japan. Email: [email protected]">[email protected]References
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