Abstract

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.

Highlights

  • IntroductionChinese Hamster Ovary (CHO) cells are the heavy-duty workhorse in the biopharmaceutical industry [1]

  • For over three decades, Chinese Hamster Ovary (CHO) cells are the heavy-duty workhorse in the biopharmaceutical industry [1]

  • When lentivirus was successfully constructed, its titer value was estimated based on the supplementary Fig 1 where the fluorescence rate was about 20% and the titer was ~108 TU/ml based on formula mentioned in method part. 3.2 Highly expressed model cell line construction and identification of insert’s site

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Summary

Introduction

Chinese Hamster Ovary (CHO) cells are the heavy-duty workhorse in the biopharmaceutical industry [1]. Establishing CHO expressing cell line through traditional methods is time-consuming and labor-intensive work mainly because of the unwanted phenotypic heterogeneity caused by position effect [5, 6]. Position effect which refers to the influence of the chromosomal location of a gene on its activity was proposed for over 90 years [7]. Position effect was the main reason causing the instability issue in traditional cell line construction process [6]. Site-specific integration (SSI) of transgenes into stable hot spots, were generally considered to be able to overcome such phenotypic heterogeneity caused by position effect and could maintain long-term expression stability [1, 3, 6, 10, 11]

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