Chromosomal Manipulation by Site-Specific Recombinases and Fluorescent Protein-Based Vectors
Figure 7
Effect of augmented Cre activity on inversion frequency.
A, Representative clones for each loxP interval (NSC27 for 1.1 Mb, NSF331 for 6.1 Mb, NSG118 for 10.5 Mb, and NSI95 for 39.8 Mb) were infected with a retrovirus expressing nuclear localization signal (NLS)-tagged Cre and puromycin-resistance gene. Simultaneously, tamoxifen (OHT) was added to activate MerCreMer. One day after infection, 0.25 µg/ml puromycin was added to select infected cells. Retrovirally Cre-transduced cells (open diamond) were analyzed by flow cytometry on day 11 to measure the frequency of fluorescence-positive cells. As a control, non-infected cells (red circle) were stimulated with OHT. B, Western blotting for retrovirally transduced Cre and MerCreMer proteins in NSC27 and NSMyc23 cells, the latter of which is clone #23 used for MYC/IgH translocation induction (Fig. 2B–D and Fig. S2). Retrovirus-infected cells were maintained in the presence of puromycin more than two weeks before lysate preparation.