Inducible and reversible expression and knockdown from the Col1a1 locus using the FLP-Inducer system.

<p>(a) Schematic representation of Col1a1-FLP-In targeting strategy. Electroporation of pFLP-Inducer-dsRed-shGL3 and pFLPo recombinase into mouse embryonic stem (mES) cells pre-targeted with Col1a1 FRT Hygromycin (–ATG) (promoter) enables gain of Hygromycin resistance and site specific integration of the FLP-Inducer construct. (b) Southern Blot of two mouse embryonic stem cell lines containing Col1a1-PGK-FRT-neo-FRT (–ATG) Hygro targeted to the <i>Col1a1</i> locus and two G418 resistant WT lines, using a 5’ internal probe. (c) dsRed microscopy of a Col1a1-FLP-Inducer- dsRed-shGL3 Hygromycin-resistant mES cell line in the presence (right panel) or absence (left panel) of 1μg/ml Doxycycline for 24hrs. (d) FACS analysis of parent mES Col1a1 FRT Hygromycin (–ATG) (promoterless) cells (Orange) or mES Col1a1-FLP-Inducer- dsRed-shGL3 cells (blue and red) in the presence (red) or absence (blue and orange) of 1μg/ml Doxycycline for 24hrs. (e) Median dsRed fluorescence in FACS analysis in (d). (f) shRNA mediated knockdown of P53 from the Col1a1-FLP-Inducer mES cells. Col1a1-FLP-Inducer-dsRed-shGL3 or Col1a1-FLP-Inducer-dsRed-shP53 were incorporated into the Col1a1 locus in mES cells by recombination mediated cassette exchange (RMCE) and shRNA induced using 1μg/ml Doxycycline, then allowed to recover by Doxycycline removal for indicated periods. P53 and tubulin protein expression were determined by immunoblot.