Generation of Mtf1 conditional knockout mice and liver-specific deletion
Mtf1 conditional knockout mice were generated in collaboration with Dr Michael Leviten (San Carlos, CA). Two genomic clones containing exons 3 to 6 of Mtf1 were used to construct a gene targeting vector for homologous recombination (Supplementary Data). A neomycin resistance cassette (PGK-neo) flanked by two loxP sites was cloned into the SacI site 5′ of exon 3 of Mtf1, the third loxP site was cloned into the ScaI site 3′ of exon 4. A thymidine kinase (TK) cassette was inserted in the HpaI site 3′ of exon 6. 129 ES cells were electroporated with the linearized targeting vector, selected in the presence of G418 and FIAU, and screened for correct integration events by PCR and Southern blot analysis. Transient expression of Cre recombinase led to removal of the PGK-neo cassette, and mice carrying the modified Mtf1loxP allele were generated by injection of positive clones into C57Bl/6 blastocysts and subsequent crosses. Homozygous conditional knockout animals (Mtf1loxP/loxP) were crossed with the Cre recombinase transgenic line Mx-cre (a gift from Prof. Michel Aguet) to obtain an inducible, liver-specific Mtf1 knockout line. The mice were genotyped by PCR using the following primers (Microsynth): Cre recombinase: 5′-CTATCCAGCAACATTTGGGCCAGC-3′; 5′-CCAGGTTACGGATATAGTTCATGAC-3′, Mtf1loxP or wild-type allele: 5′-CACACCCAGTTTGTGTATGTCTTC-3′; 5′-CAGTCACAAGCAAATTACCAAACACTGCC-3′.