Discovery of a stable expression hot spot in the genome of Chinese hamster ovary cells using lentivirus-based random integration

The conventional method for construction of stable expression cell lines is mainly based on random integration. However, one drawback of random integration is that the target gene might be integrated into a heterochromatin region or an unstable region of chromatin, thus requiring multiple rounds of selection to obtain desirable expressing cell lines. Rational cell line construction can overcome this shortcoming by integrating transgenes specifically into a stable hot spot within the genome. As such, the discovery of novel effective hot spots becomes critical for this new method of cell line construction. Here we report a practical method for discovery of new stable hot spots through random integration of lentivirus. We describe the thorough study of a hot spot located at NW_006880285.1. The expression stability of this hot spot was verified by detecting Zsgreen1 reporter gene expression for over 50 passages. When cells were adapted to suspension culture, they continuously expressed the Zsgreen1 reporter gene. In addition, this cell suspension was able to stably express the reporter gene for an additional 50 passages. Another finding was that cells with the NGGH gene inserted into the same hot spot were also able to stably express respective protein over 50 passages. In summary, this research offers an easy and new method for researchers to identify stable hot spots within the Chinese Hamster Ovary (CHO) genome on their own, thus contributing to the development of site-specific integration studies in the future.