Generation of Dusp8 KO and transgenic mice

A vector targeting the catalytic domain of DUSP8 was generated to replace exons 5 and 6 of the Dusp8 gene with a neomycin cassette, which was electroporated into mouse SV129j-based embryonic stem (ES) cells. Correctly targeted ES cells were identified by Southern blotting and subsequently injected into C57Bl/6 blastocysts to generate chimeric mice, then germ line and homozygous Dusp8 null mice in a final C57Bl/6-SV129j background. The following PCR primers were used to genotype WT and KO mice. WT: forward, 5’-tgggcatgtcttctgacgac-3’, reverse, 5’-agtgaggtccatcagtctgc-3’; KO: forward, 5’-ctccaccatgccctcttc-3’, reverse, 5’-gcgcatcgccttctatcgc-3’. To generate DUSP8 inducible transgenic mice, a cDNA encoding this protein was cloned into SalI and HindIII sites of the modified murine α-myosin heavy chain (αMHC) promoter expression vector (Dr. Jeffrey Robbins, Cincinnati Children’s Hospital Medical Center) to allow for doxycycline-regulated expression of the DUSP8 transgene in the presence of a cardiac-specific tetracycline transactivator (tTA)-containing transgene. DUSP8 transgenic mice were genotyped using the following primers: forward, 5’-gggaagtggtggtgtaggaaag-3’, reverse, 5’-tttagggcaggagttgctgg-3’. Doxycycline (625 mg/kg) was administered in the food until weaning. Mice were then switched to regular chow diet for an additional 6 weeks to allow DUSP8 expression.¹⁴ All animal breeding and experimentation were approved by Cincinnati Children’s Hospital Medical Center Institutional Animal Care and Use Committee.