Deletion of Trsp gene leads to increased oxidative stress and induction of Nrf2-target genes in hematopoietic tissues. (A) The targeting strategy for Trsp gene by the Cre-loxP recombination system. Trsp gene is flanked with 2 loxP sequences, designated by closed arrowheads. Arrows indicate the position of primers used for PCR analysis in B. (B) PCR analysis of Trsp^fl/del and Trsp^fl/del:Mx1-Cre mice after the pI-pC treatment. Fragments derived from floxed allele (494 bp; filled triangles) and recombined allele (465 bp; open triangles) were amplified by SeC1F and SeC1R primer pair and SeC1F and SeC2R primer pair, respectively. (C) Expressions of GPx1 protein in Trsp^fl/del and Trsp^fl/del:Mx1-Cre mice on an Nrf2^+/+ or Nrf2^–/– background were examined by immunobloting. Note that the levels of GPx1 protein were significantly decreased in hematopoietic organs of Trsp^fl/del:Mx1-Cre mice, although those in livers were preserved. Nonspecific bands are indicated by asterisk. (D-E) Expressions of GPx1 (D), NQO1, GCLC, HO-1, and TXNRD1 (E) mRNA in whole bone marrow cells from mice of each genotype were examined by real-time RT-PCR. Ribosomal 18S subunit was used as an internal control. (F) ROS levels in whole bone marrow cells of Trsp^fl/del and Trsp^fl/del:Mx1-Cre mice on an Nrf2^+/+ (top panel) or Nrf2^–/– background (bottom panel) were evaluated by flow cytometry using the redox-sensitive dye H₂DCFDA.