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. Author manuscript; available in PMC: 2014 Jul 8.
Published in final edited form as: Nat Methods. 2014 Jun 8;11(7):763–772. doi: 10.1038/nmeth.2996

Figure 5. Exclusion logic using INTRSECT.

Figure 5

a) Schematics representing target populations of cells expressing Cre and Flp (left) by nesting Cre-dependent directional control of a central exon and Flp-dependent directional control of all three exons (right). b) Intermediate states driven by Cre and Flp on constructs dependent on the exclusive presence of a single recombinase for functional expression (dashed box). c) cDNA prepared from mRNA of HEK293 cells transfected with indicated constructs and recombinases and source DNA were amplified by PCR with primers in exons 1 and 3 and separated by gel electrophoresis. Note variation in the size of bands from intron-containing DNA template and uniformity of cDNA bands, suggesting successful splicing. d) Sequences of bands from b. Yellow denotes differences from ChR2-eYFP map sequence, representing intron sequence. e,f) eYFP is only expressed in cultured neurons transfected with combinations of Cre (blue), Flp (red), and Con/Foff (e; green) or Coff/Fon (f; green) in the exclusive presence of Cre or Flp. g) Dot exemplar (top) and replicate histograms (bottom) of flow cytometry analysis of HEK293 cells transfected with indicated recombinase/construct combinations indicates expression in the presence of a single recombinase (arrowheads) that is extinguished when both recombinases are present, which was confirmed by Gaussian mixture model analysis (multiple populations fit in 85%-96% of matched single-recombinase transfections, single population fit in 94%-99% of double-recombinase transfections). h,i) Whole cell photocurrents (h) and action potentials (i) recorded from primary neurons transfected with ChR2-eYFP or Con/Fon, Con/Foff, Coff/Fon-ChR2-eYFP and Cre and Flp as indicated. All error bars s.e.m.