Article Title: High-performance chemical and light-inducible recombinases in mammalian cells and mice
Figure Lengend Snippet: Swapping chemical-inducible dimerization domains yield a large repertoire of small-molecule inducible recombinases. ( a ) A Signal-to-Noise (SNR) metric can be used to capture distinguishability of “on” and “off” states, accounting for both the absolute difference in mean signal expression and spread (“noise”) of the distributions. SNR dynamics in units of decibels (dB) were captured over 100 hours ( b ) post-transfection for particular amino acid splits of inducible Cre, Flp and PhiC31 systems incorporating the gibberellin (GIB), rapalog (RAP) and abscisic acid (ABA) dimerization domains GAI/GID1, FKBP/FRB and PYL/ABI, respectively. A 4-hydroxytamoxifen (4OHT)-inducible pCAG-ER T2 -Cre-ER T2 construct is also included in ( b ). Various splits of Cre ( c and d ) Flp ( e and f ), VCre ( g and h ), PhiC31 ( i and j ) TP901 ( k and l ) and Bxb1 ( m and n ) were incorporated with the GIB-, RAP- and ABA-associated dimerization domains. Measurements of GFP mean fluorescence intensity (M.F.I.) in units of molecules of equivalent fluorescein (MEFL) and SNR were made for plus drug (colored blue, purple, and pink squares corresponding to gibberellin, rapalog, and abscisic acid added two hours post-transfection) and minus drug (white squares) conditions. Constitutive recombinases (black squares) and blank vectors (gray squares) were also transfected as a comparison. Red diamonds indicate SNR score. Error bars of M.F.I. indicate the geometric standard deviation of means for three transfected cell cultures.
Article Snippet: Small-molecule chemical induction1000X stocks of abscisic acid (100mM, Gold Biotechnology Cat. No. A-050-500) and gibberellin ester (10mM, Toronto Research Chemicals Cat. No. G377500) were made in 100% ethanol and stored at −20°C.
Techniques: Expressing, Transfection, Construct, Fluorescence, Standard Deviation