Fig. 6: RAG-mediated recombination of split-GFP construct generates diversity—up to three colors in vivo.

a Diagram of the pY112-CJCG-ES-H20 plasmid. The first portion of GFP is flanked by a 12-RSS followed by an intersignal region and then two modules each consisting of a 23-RSS, a gene fragment that creates eGFP or Sapphire, and a terminator. Triangles represent RSSs. Each gene fragment has 20 bp of homology to the first portion of GFP. Due to the 12/23 rule of RAG recombination, there are only two expected resolutions to recombination, one which creates eGFP and another which creates Sapphire. b Expected phenotype change for cells bearing the pY112-CJCG-ES-H20 plasmid as recombination is induced. Initially, all cells are non-fluorescent, but RAG-mediated recombination leads to subpopulations with eGFP or Sapphire fluorescence. c 2-color recombination of cells with target plasmids that have a fragment order of eGFP then Sapphire (pY112-CJCG-ES-H20) or Sapphire then eGFP (pY112-CJCG-SE-H20). d 2-color recombination with fragment order eGFP then Azurite (pY112-CJCG-EA-H20). e 3-color recombination using a target plasmid with a fragment order of eGFP, Sapphire, then Azurite (pY112-CJCG-ESA-H20). For all tests, cells were cultured for 9-d in SG-Leu media prior to analysis with flow cytometry. BY4742 are wild-type cells that do not contain V(D)J recombination genes. In (c–e) data are presented as mean values ± SD; n = 3 biological replicates. Statistical significance was calculated with a two-way ANOVA with repeated measures across colors and Fisher LSD test (for c and d) or Tukey test (for e) (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). From left to right, the highlighted p values are, in (c), p = 0.0027, 0.1741, 0.0001, and 0.0001; in (d), p = 0.0012 and < 0.0001; and in (e), p = 0.0327, 0.8738, and 0.0136. Source data are provided in the Source Data file.