LoxP recognition sites were inserted around the second exon of the murine Esm1 gene via homologous recombination in the ES cells. A murine cDNA fragment containing the coding sequence from exons 2 and 3 followed by 2 copies of the bovine growth hormone polyadenylation signal sequence was inserted in frame, and downstream of the loxP-flanked area, the targeting construct also contained a beta-galactosidase (lacZ) reporter cassette, which carried an artificial splice acceptor and an internal ribosomal entry site (IRES) at the 5â-end and the SV40 polyadenylation signal sequence at the 3â-end. The targeting construct also contained a neomycin resistance cassette surrounded by Frt sites allowing Flp recombinase-mediated removal. Through interbreeding with PGK-Cre deleter mice, exon 2 of the Esm1 gene was globally deleted. No beta-galactosidase reporter activity was detectable in heterozygous or homozygous mutants, and it was subsequently confirmed that Esm1-lacZ fusion transcripts were not detectable by qPCR.