Lman1 mutant alleles. (A) The Lman1 conditional gene-trap allele (Lman1^cgt) contains a gene-trap insertion in intron 1 flanked by 2 FRT sites. Mice carrying this allele were crossed to β-actin FLP transgenic mice. Mice heterozygous for the resulting Lman1 floxed allele (Lman1^fl) were crossed to Alb-Cre⁺ or Tek-Cre⁺ transgenic mice to excise exons 2 and 3, generating the Lman1 null allele (Lman1⁻) in selected tissues. Gray blocks represent exons. A, B, C, D, and E represent genotyping primers. (Adapted from the Knockout Mouse Project. General conditional gene targeting scheme: https://www.komp.org/alleles.php#conditional-promoter; Lman1 targeting: https://www.komp.org/geneinfo.php?geneid=66654); cgt, conditional gene trap; fl, floxed; En2 SA, splice acceptor of mouse En2 exon 2; IRES, encephalomyocarditis virus (EMCV) internal ribosomal entry site; lacZ, Escherichia coli β-galactosidase gene; pA, SV40 polyadenylation signal; βact:neo, human β-actin promoter-driven neomycin cassette. (B) A 3-primer PCR assay (primers C, D, and E) distinguishes the Lman1⁺ allele (434 bp) from Lman1^cgt (565 bp). (C) PCR genotyping assay used for tail-snip genomic DNA from a Lman1^fl/fl, Alb-Cre⁺ mouse, with the addition of a liver biopsy genomic DNA sample from the Lman1^fl/fl, Alb-Cre⁺ mouse to demonstrate excision of Lman1 exons 2 and 3 in the liver. A 3-primer PCR assay (primers A, B, and C) distinguishes the Lman1⁺ (444 bp), Lman1^cgt (508 bp), Lman1^fl (508 bp), and Lman1⁻ (635 bp) alleles. Alb-Cre primers were used to determine the Cre genotype of offspring from matings of Lman1^fl/+, Alb-Cre⁺ mice with Lman1^fl/fl mice. Comparison of the signal in DNA from liver tissue of an Lman1^fl/fl, Alb-Cre⁺ mouse (lane 7) to tail DNA from the same animal (lane 6) indicates a high degree of specific excision in the liver. A similar PCR genotyping strategy of tail-snip genomic DNA was used to identify Lman1^fl/fl, Tek-Cre⁺ mice, with primer trio A/B/C demonstrating the Lman1 genotype and with Tek-Cre–specific primers rather than Alb-Cre primers (not shown). (D) Western blot of spleen, heart, liver, pancreas, lung, and kidney tissues from a wild-type C57BL/6J mouse and a Lman1^fl/fl, Alb-Cre⁺ (hepatocyte-knockout) mouse, demonstrating nearly complete and tissue-specific Lman1 knockout in the liver of Lman1^fl/fl, Alb-Cre⁺ mice. The lower-molecular-weight band observed in the heart samples represents a nonspecific band due to cross-reactivity with the LMAN1 antibody, because this same band is observed in the heart tissue of ubiquitous Lman1 knockout mice (not shown).