Strategy employed for the inactivation of the murine ferritin light and heavy genes.

<p>For the inactivation of the <i>Ftl</i> gene, exons 1 and 2 were deleted (A). The deletion targeting vector contained the <i>MC1tk</i> (thymidine kinase) expression cassette, 5.72 kb of 5’ <i>Ftl</i> homology, a <i>pGK-neo</i> (neomycin-resistance gene) expression cassette, 0.83 kb of the <i>Ftl</i> gene containing exons 1 and 2, and 5.74 kb of 3’ <i>Ftl</i> homology. Three loxP sites were introduced by targeted mutagenesis between the <i>neo</i> cassette and after exon 2 of <i>Ftl</i>. Homologous recombination in ES cells identified by Southern blot hybridization analysis using the external probe A after digestion with XhoI. The wild-type allele has 8.03 kb and the floxed allele has 7.30 kb (B). Germline chimeras were crossed with C57Bl/6 females, and mice carrying the floxed <i>Ftl</i> (<i>Ftl</i>^<i>f/+</i>) allele were then crossed with mice expressing the <i>Cre</i> recombinase. Mice showing the deletion of exons 1 and 2 of <i>Ftl</i> and the <i>neo</i> cassette were crossed with C57Bl/6 mice to remove the <i>Cre</i> allele. Mice were maintained in C57Bl/6 background and the presence of the <i>Ftl</i> allele determined by PCR, resulting in a 180-bp fragment for the wild-type allele and in a 204-bp fragment for the knock-out allele (B). The four exons of the mouse <i>Fth</i> gene were targeted for deletion (C). The deletion targeting vector contained the <i>MC1tk</i> expression cassette, 4.44 kb of 5’ <i>Fth</i> homology, a <i>pGK-neo</i> expression cassette, 2.4 kb of the <i>Fth</i> gene containing all four exons, and 5.0 kb of 3’ <i>Fth</i> homology. Three loxP sites were introduced by targeted mutagenesis between the neo cassette and after exon 4 of <i>Fth</i>. Homologous recombination in ES cells identified by Southern blot hybridization analysis using the external probe A after digestion with HindIII. The wild-type allele has 6.25 kb and the floxed <i>Fth</i> (<i>Fth</i>^<i>f/+</i>) allele has 5.62 kb (D). Germline chimeras were crossed with C57Bl/6 females, and mice carrying the floxed <i>Fth</i> allele were then crossed with mice expressing the <i>Cre</i> recombinase. Mice showing complete loss of <i>Fth</i> and the <i>neo</i> cassette were crossed with C57Bl/6 mice to remove the <i>Cre</i> allele. Mice were maintained in C57Bl/6 background and the presence of the <i>Fth</i> allele determined by PCR, resulting in a 252-bp fragment for the wild-type allele and in a 354-bp fragment for the knock-out allele (D). The symbol ► represents the loxP sites; f, floxed alleles.