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. 2015 Jun 2;290(30):18343–18360. doi: 10.1074/jbc.M115.664490

FIGURE 2.

FIGURE 2.

Generation of a conditional Dhs knock-out mouse strain using ES-cell clones harboring a targeted mutation of the Dhs gene. A, schematic representation of the strategy for achieving a knock-out of the Dhs gene. The seven exons designed for deletion are depicted in a lighter color. Blue arrows indicate regions encoding amino acids that are essential for binding of NAD; green arrows indicate regions essential for binding of spermidine (86). The targeted mutation of the Dhs gene consists of a lacZ-reporter cassette (SA, splice acceptor; IRES, internal ribosome entry site; lacZ, β-galactosidase gene; pA, polyadenylation signal), a neomycin-selection cassette (PβAct, eukaryotic β-actin promotor; neoR, neomycin resistance gene), and three loxP and two FRT sites. B, genotyping strategy for Cre-mediated knock-out of the Dhs gene. Animals harboring the floxed Dhs genes were mated to Cre-deleter mice (B6.C-Tg(CMV-cre)1Cgn/J strain or B6.Cg-Tg(CAG-cre/Esr1+)5Amc/J113 strain plus oral 4-OHT administration. Usage of three oligonucleotides (Dhs-3′-arm, Dhs-for, and Dhs-rev) allows simultaneous detection of all possible alleles (+, p, and −), including the deleted allele (−), which is achieved by Cre-mediated recombination.