In situ conversion of defective Treg into SuperTreg cells to treat advanced IPEX-like disorders in mice

摘要

Strategy for GFP-labeled, Treg-specific reversible knockout. This method requires a conventional -floxed allele ( ) paired with a multifunctional reversible KO (ΔR) allele (top left), and sequential action of Cre and Flpo recombinases (middle and bottom). Depicted is the status of the alleles (left) and the corresponding protein expression patterns (right). Note that Cre was expressed from the endogenous FoxP3 locus located on the X chromosome subject to random inactivation, and so the reversible KO (rKO) mice carried either one or two alleles depending on the sex. SA splicing acceptor, Neo neomycin-resistance gene, FRT flippase-recognition target (red dot). The alleles. The gene-trap cassette in Δ is inserted after E8 in the locus, and the floxed exons in highlighted in pink. Also depicted are the homology arms used to make the targeting construct for generating Δ , and the PCR primers for genotyping. – Characterization of mouse samples. Shown are the representative results from two biological replicates. The tail from a mouse was subjected to PCR analysis using primer pairs a/b and c/d (depicted in ) to verify successful targeting ( ); GFP and GFP Tregs isolated from TAM-treated rKO mice were analyzed by PCR and RT-PCR to detect the excision of the gene-trap cassette ( ) and restoration of expression ( ), respectively. The control mouse in has the same genotype as rKO, except that it carried instead of . Kinetics of GFP loss following TAM administration. TAM (full dose) was given via oral gavage, and GFP expression in Tregs and conventional CD4 cells in tail blood monitored by FACS. The control mouse did not carry .