Figure 1.
DT40 B lymphocytes transfected with a reversible switching construct and expressing mer-Cre-mer show 4-hydroxytamoxifen independent Cre activity.
A Schematic of the “Flip” construct. Exon 4 of NUDT9 was replaced with a CMV promoter, an in-frame region (containing a NUDT9 cDNA and an inverted mCherry cDNA, each followed by a poly-A region) flanked by loxp sites, and a neomycin selection cassette. Treatment of cells stably expressing the construct with 4-hydroxytamoxifen (OHT) should lead to reversible flipping of the floxed region, as shown in the cartoon, and subsequent expression of mCherry red fluorescent protein in ∼50% of the cells. B Stable integration of the Flip construct was verified in clone #27 via genomic PCR. The positive control represents a different construct that could be amplified with the same primer set. C Following stable transfection of clone #27 with a mer-Cre-mer (mCrem) expressing vector, cells from various clones were lysed and 50 µg of protein from each were run on an 8% SDS-PAGE gel. mCrem was visualized with polyclonal rabbit anti-Cre antibody (1∶2000, Novagen), and a highly expressing clone, 27Flip/Cre20 was selected for further study (red circle). D 27Flip and 27Flip/Cre20 cells were analyzed by flow cytometry, as indicated. In contrast to the 27Flip parental line, the mCrem expressing 27Flip/Cre20 cells showed robust red fluorescence in a large subpopulation despite the absence of the mer- ligand OHT.
Figure 2.
Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells is a stable, ongoing process.
mCherry (+) and mCherry (−) 27Flip/Cre20 cells were separated by FACS and grown in culture for ∼7 weeks. During this time period, a steady decrease in the purity of the sorted populations was observed by flow cytometry. X-axis, mCherry fluorescence, Y-axis, cell number.
Figure 3.
Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells correlates with mer-Cre-mer expression level.
A Following stable transfection of 27Flip cells with an mCrem expressing vector, 5 clones expressing various amounts of mCrem were selected. Cells from the clones were lysed and 50 µg of protein of each were run on an 8% SDS-PAGE gel. MCrem was visualized with polyclonal rabbit anti-Cre antibody (1∶2000, Novagen), with β-actin visualized with a polyclonal mouse antibody (1∶40000, Sigma) as a loading control. 27Flip/Cre15 and -20 were designated “high expressors”, and 27Flip/Cre26, -31, and -35 were designated “low expressors”. B 27Flip/Cre clones were either left untreated or treated with 1 µM OHT for 48 hours and then allowed to grow in culture for 18 days: all clones were subsequently analyzed by flow cytometry. The 27Flip parental cell line was used as a negative control.
Figure 4.
OHT-dependent flipping of the floxed cassette saturates by 10 nM OHT.
27Flip/Cre clones were either left untreated or treated with 1, 10, or 100 nM OHT for 48 hours and then allowed to grow in culture for 5 days from the onset of OHT treatment: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. X-axis, mCherry fluorescence, Y-axis, cell number.
Figure 5.
Efficiency of OHT-dependent flipping of the floxed cassette correlates with duration of OHT exposure in low mCrem expressors.
A 27Flip/Cre clones were either left untreated or treated with 1 nM OHT for 24, 10, 5, 2, or 1 hours, and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. B 27Flip/Cre31 cells were either left untreated or treated with 100 nM OHT for 1, 2, 5, 10, 30, 60, 120, or 240 minutes and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. X-axis, mCherry fluorescence, Y-axis, cell number.