Establishing Isogenic Inducible Cell Lines Using Founder Reporter Lines and Recombinase-Mediated Cassette Exchange

Abstract

Manipulating gene expression in mammalian cell lines is one of the most widely used methods for studying gene function. Tetracycline- and doxycycline-inducible systems are sensitive, reproducible, relatively inexpensive, and proven to work well in both cell lines and mouse models. However, obtaining homogeneous transgene expression or uniform knockdown by short hairpin RNA requires time-consuming and labor-intensive single-cell cloning to derive stable cell lines. For this reason, Tet-inducible cell systems have yet to be widely adopted. Here we describe the XT-cell method, a novel system for establishing isogenic inducible cell lines using founder reporter lines and recombinase-mediated cassette exchange. We demonstrate that, using this XT-cell method, isogenic stable Tet-inducible cell lines can be efficiently created with much less effort and time as compared with conventional methods. The XT-plasmids and the XT-founder cell lines will be a valuable resource to researchers interested in versatile modulation of gene expression in cell culture systems, and this method has the potential to expedite many aspects of biomedical research.

Keywords: :

• Tet-inducible cell line
• fluorescence founder reporter line
• RMCE
• XT-method

Author contributions

BG designed and performed experiments, analyzed data, and wrote the manuscript. TM performed cloning for some of the XT plasmids. TLW and IMS wrote the manuscript.

Acknowledgments

We thank Yuri Voziyanov for the generous gift of the pFIC plasmid. This work was supported by the National Institutes of Health [R21CA165807, RO1CA103937, RO1CA129080, and RO1CA148826], and the Ann Schreiber Research Training Programs of Excellence grant [POE/JHU/01.12] awarded to B.G. by the Ovarian Cancer Research Fund and the Tell Every Amazing Lady Louisa M. McGregor Ovarian Cancer Foundation. This paper is subject to the NIH Public Access Policy.

Competing interests

BG, IMS and TLW filed an invention disclosure for the XT-cell method.

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.tandfonline.com/doi/suppl/10.2144/000114098

Additional information

Funding

We thank Yuri Voziyanov for the generous gift of the pFIC plasmid. This work was supported by the National Institutes of Health [R21CA165807, RO1CA103937, RO1CA129080, and RO1CA148826], and the Ann Schreiber Research Training Programs of Excellence grant [POE/JHU/01.12] awarded to B.G. by the Ovarian Cancer Research Fund and the Tell Every Amazing Lady Louisa M. McGregor Ovarian Cancer Foundation. This paper is subject to the NIH Public Access Policy.