Intronic PTEN cRSS mediate RAG recombination events. (A) Top panel: Linear representation of the GFPi reporter construct that results in the inversion of GFP coding sequence during RAG-mediated recombination, and consequent GFP expression. The inverted GFP sequence (light green box) is flanked by a proximal 12-spacer RSS (light gray triangle) and a distal 23-spacer RSS (dark gray triangle) followed by the IRES-RFP as transfection control reporter (red box). GFP positivity is a measure for recombination potential. Bottom panel: Control in vitro RAG recombination assay; flow cytometry analysis of HEK293T cells transiently transfected with either an irrelevant, mock vector (in the absence of RAG1/2 expression vectors; negative control) or the GFPi-reporter construct containing the consensus 12- and 23-RSS in the presence of RAG1/2 expression vectors.²⁹  The flow cytometry plots show the expression of GFP and RFP within gated live cells defined by FSC and SSC parameters (not shown) and the values represent the percentage of each cell population in the quadrants. The gate used to discriminate RFP-positive from RFP-negative cells is depicted by a red square and used for the contour plot analysis. The efficiency of recombination is indicated as the percentage of GFP-positive (recombination positive) cells within the RFP-positive (transfected) population. (B) Flow cytometry analysis of HEK293T cells transiently transfected with the GFPi variant constructs containing specific 12-spacer cRSS (LMO2, SCL/TAL1, PTEN-cRRS1, or the Jβ2.2-RSS) site combined with the consensus 23-spacer RSS (left panel). The GFPi variant constructs containing the consensus 12-spacer RSS²⁹  were combined with 23-spacer PTEN cRSS2, cRSS3, cRSS4, or the control SCL/TAL1 23-spacer cRSS version (right panel). The human LMO2 12-spacer cRSS and the mouse Jβ2-2 bona fide RSS were used to establish the range of recombination activitites for low-efficiency RSSs as measured by the GFPi reporter assay. The 12- and 23-spacer versions of the human SCL/TAL1 cRSS were used to define the lower limit of detection of cRSS function in this reporter assay. Average percentage ± SD of GFP⁺ cells in the RFP⁺ population are derived from 4 to 5 independent experiments. (C) Recombination index was determined by normalizing the recombination efficiencies of each indicated reporter to that of GFPi Con12/23 and recombination efficiencies were calculated subtracting the GFP background of each respective unrecombined control. Values represent the mean ± SEM of 3 independent experiments with 3 replicates per condition; *P < .05; **P < .01; and ***P < .0001.