To determine the accuracy of the HygR-5′ EGFP reporter system for monitoring gene targeting efficiency, we subsequently investigated whether this reporter system could reproduce previous findings of gene targeting efficiency achieved with various types of targeting vectors. We first infected (as an AAV-based targeting vector) or transfected (as a plasmid-based targeting vector) HCT116 and DLD-1-derived HygR-5′ EGFP reporter clones with ATG-less TV and then selected the cells with G418. Flow cytometric analyses demonstrated that AAV-based ATG-less TV infection achieved an average of >100-fold gene targeting efficiency compared with that of the plasmid version of ATG-less TV (Figure 4A), reproducing previous results (5, 6, 16). We also addressed the impact of the promoter-trap strategy on gene targeting efficiency in this system. To this end, we constructed an AAV-based targeting vector similar to ATG-less TV, except that it lacked a synthetic intron and IRES, containing a poly(A) site and an SV40 promoter to drive NeoR instead, thus no longer supporting the promoter-trap strategy (SV40p TV; Figure 1). Fluorescence flow cytometric analyses demonstrated that the infection of HygR-5′ EGFP reporter cell clones with ATG-less TV achieved, on average, 40-fold greater gene targeting efficiency than SV40p TV (Figure 4B), which is similar to the results reported by previous studies (6, 16). Increasing homology length is also likely to increase targeting rates (11). We thus shortened the 5′ homology arm of ATG-less TV and examined its impact on gene targeting efficiency. The 5′ homology arm of ATG-less TV is 719 bp in length, and a 113-bp fragment at its 5′ terminus was deleted to create another targeting vector, 3′ EGFP TV (Figure 1). This truncation resulted in a reproducible decrease in gene targeting efficiency, although our results did not reach statistical significance (Figure 4C). Overall, our HygR-5′ EGFP reporter system successfully reproduced the impact of targeting vector designs on gene targeting efficiency described in previous studies.
To clarify the advantage of the use of our HygR-5′ EGFP reporter system in monitoring gene targeting efficiency, we next compared the efficiency of generating reporter clones in our HygR-5′ EGFP system with that of a conventional reporter system that uses an unfused 5′ EGFP gene as a reporter (5′ EGFP reporter). We first isolated multiple HCT116-derived cell clones transduced with the 5′ EGFP reporter vector and infected the clones with ATG-less TV. The resulting bulk population of G418-resistant cells from each 5′ EGFP reporter clone was then analyzed by fluorescence flow cytometry, similar to that for the HygR-5′ EGFP reporter clones. It appeared that functional reporter clones were obtained with higher efficiency by the HygR-5′ EGFP reporter system than that with the 5′ EGFP reporter system (Table 1), although the difference did not reach statistical significance presumably because of the limited number of cell clones analyzed. Southern blot analysis demonstrated that the two functional 5′ EGFP reporter clones are likely to carry single 5′ EGFP reporter vectors within the genome (data not shown).
We also addressed the ability of reporter cell clones in the HygR-5′ EGFP and 5′ EGFP reporter systems to monitor gene targeting efficiency over time. Reporter clones bearing functional HygR-5′ EGFP or 5′ EGFP reporter genes were maintained in culture for long durations (no less than 4 weeks), infected with ATG-less TV, selected with G418, and then analyzed by fluorescence flow cytometry. GFP-positive ratios in individual reporter clones were then compared with those obtained before the above mentioned long-term culture. We found that GFP-positive ratios before and after the long-term culture were equivalent in the HygR-5′ EGFP reporter clones; however, in 5′ EGFP reporter clones, GFP-positive ratios were dramatically decreased after the long-term culture (Figure 5). These data demonstrated that our HygR-5′ EGFP reporter system provides a prolonged capacity to monitor gene targeting efficiency relative to a conventional reporter system.