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

Figures & data

Figure 1. The XT-cell method.

(A) Diagram of plasmid vectors used in this study. (B) The steps of the XT-method. Hygromycin-resistant and puromycin-sensitive XT founder reporter cell lines that show optimal tetracycline induction were established in the first step. A donor vector including the sequence of interest (SOI) was transfected with pFIC (expressing Flp and Cre recombinases) into the XT reporter cells and selected by puromycin. The resulting puromycin resistant cells are due to successful recombinase-mediated cassette exchange (RMCE) and retain tetracycline induction capability as the XT founder reporter cells.

Figure 2. The XT-method efficiently establishes inducible cell lines.

(A) mCherry expression in HeLa-XT1 mCherry cells were analyzed using flow cytometry, in the absence or presence of doxycycline. (B) GFP expression was analyzed in HeLa-XT1 GFP cells that arise from RMCE. Doxycycline (0.5 µg/mL) was added in culture media for 2 days before flow cytometry analyses. (C) Western blots showing ARID1A expression in the HeLa-XT1 ARID1A cells 2 days after 0.5 µg/mL doxycycline induction. (D) Western blots showing ARID1A expression in the HeLa-XT1 shARID1A cells 2 days after 0.5 µg/mL doxycycline induction. GAPDH serves as a loading control. Note that the ARID1A signal in (D) was overexposed.

Table 1. Optimization of RMCE efficiency by altering the amount of recombinases expressed.

Figure 3. Flowchart of the XT-cell method.

The current procedures for establishing founder XT cells are time-consuming and labor intensive. However, after creating founder lines through collaborative efforts, these cell lines can be used to establish doxycycline-inducible cells containing the sequence of interest quickly with minimal hands-on time.