FIG. 1.

Gene targeting of Txnrd1. (A) Schematic drawing of mouse Txnrd1 3′ region including exons 12 to 15. The last exon harbors the coding region for the redox center located at the C terminus, including the Sec codon UGA (marked with an asterisk), the SECIS element, the AU-rich elements, and the endogenous transcription termination signal. The bottom panels depict the conditional gene targeting strategy to flank exon 15 with loxP sites (black triangles) (floxed). The neo cassette is flanked by frt sites (open triangles). TK, thymidine kinase. The Cre-mediated deletion of the floxed Txnrd1 allele led to the inactivation of Txnrd1. Expected DNA fragments after characteristic restriction digests (V, EcoRV; A, AseI) detected with corresponding 5′ and 3′ external probes (hatched rectangles) are drawn schematically. (B) Homologous recombination of the targeting construct in ES cells was verified by Southern blotting with both 3′ (I) and 5′ (II) external probes. (C) Germ line transmission of the conditional Txnrd1 allele was tested by PCR (the binding regions of the specific primers are marked with arrows in panel A). Panel I, the floxed Txnrd1 allele gives rise to a 64-bp-longer product than the WT allele; panel II, genotyping by Southern blotting of hemizygous KO mice obtained by breeding of floxed Txnrd1 mice with Cre deletion mice; panel III, genotyping of embryos isolated at E9.5 from hemizygous Txnrd1 intercrosses. (D) RT-PCR analysis with mRNAs isolated from E9.5 embryos of each genotype as templates. The primer pair Oligo Txnrd1-E13f and -E15r indicated the absence of exon 15 in Txnrd1 KO embryos. Very low levels of a truncated Txnrd1 message could be detected in Txnrd1 KO embryos by use of the primer pair Oligo Txnrd1-59 and -60 specific for the central region of Txnrd1.