Figure 1.
Figure 1. Targeted disruption of the mouse melanotransferrin (MTf) gene. (A) Targeting strategy used for deletion of mouse MTf exons 2, 3, and 4 and reversal of part (443 bp) of the MTf promoter region and the translation initiation codon. The genomic structure of the wild-type MTf allele is shown at the top depicting the promoter region, 16 exons, and intervening introns. Primers used for identification of the wild-type allele are denoted P1 and P2, and fragments used for Southern analysis are denoted as the 5′ and 3′ probes. The targeting construct is shown below the wild-type allele. In this construct, the 1.4-kb BamHI fragment containing exon 1, intron 1, and part of the promoter region is reversed. After homologous recombination, the neomycin resistance (neor) targeted allele contains an additional NheI restriction enzyme site introduced with the neor gene cassette (Neo allele). In the Neo allele, exons 2, 3, and 4 have been replaced with the neor gene cassette flanked by lox-P sites and exon 1 is reversed. The neo cassette was deleted from the genome by mating of MTf+/– males to B6-deleter females to obtain the knockout (KO) allele. The P3 and P4 primers detect the targeted allele. (B-F) Confirmation of MTf ablation in the mouse at the genomic DNA, mRNA, and protein levels. (B) Genotype identification by Southern blot analysis from mice containing the neor cassette. (C) Genotype identification by Southern blot analysis from mice where there has been cre-mediated deletion of the neor cassette. (D) Genotype identification by PCR analysis of mice with the cre-mediated deletion of the neor cassette. P1 and P2 primers detect a wild-type fragment of 1.2 kb (first lane). Primers P3 and P4 detect a fragment of 0.7 kb in the targeted knockout (KO) allele (second lane) and fragments of 0.7 and 1.2 kb in the MTf+/– mice (third lane). (E) RT-PCR of MTf and transferrin receptor 1 (TfR1) mRNA transcripts in MTf+/+, MTf+/–, and MTf–/– mice. (F) Western analysis of MTf and TfR1 in brain and testis from MTf+/+ and MTf–/– mice. Results are representative results in a typical experiment from 3 separate experiments.

Targeted disruption of the mouse melanotransferrin (MTf) gene. (A) Targeting strategy used for deletion of mouse MTf exons 2, 3, and 4 and reversal of part (443 bp) of the MTf promoter region and the translation initiation codon. The genomic structure of the wild-type MTf allele is shown at the top depicting the promoter region, 16 exons, and intervening introns. Primers used for identification of the wild-type allele are denoted P1 and P2, and fragments used for Southern analysis are denoted as the 5′ and 3′ probes. The targeting construct is shown below the wild-type allele. In this construct, the 1.4-kb BamHI fragment containing exon 1, intron 1, and part of the promoter region is reversed. After homologous recombination, the neomycin resistance (neor) targeted allele contains an additional NheI restriction enzyme site introduced with the neor gene cassette (Neo allele). In the Neo allele, exons 2, 3, and 4 have been replaced with the neor gene cassette flanked by lox-P sites and exon 1 is reversed. The neo cassette was deleted from the genome by mating of MTf+/– males to B6-deleter females to obtain the knockout (KO) allele. The P3 and P4 primers detect the targeted allele. (B-F) Confirmation of MTf ablation in the mouse at the genomic DNA, mRNA, and protein levels. (B) Genotype identification by Southern blot analysis from mice containing the neor cassette. (C) Genotype identification by Southern blot analysis from mice where there has been cre-mediated deletion of the neor cassette. (D) Genotype identification by PCR analysis of mice with the cre-mediated deletion of the neor cassette. P1 and P2 primers detect a wild-type fragment of 1.2 kb (first lane). Primers P3 and P4 detect a fragment of 0.7 kb in the targeted knockout (KO) allele (second lane) and fragments of 0.7 and 1.2 kb in the MTf+/– mice (third lane). (E) RT-PCR of MTf and transferrin receptor 1 (TfR1) mRNA transcripts in MTf+/+, MTf+/–, and MTf–/– mice. (F) Western analysis of MTf and TfR1 in brain and testis from MTf+/+ and MTf–/– mice. Results are representative results in a typical experiment from 3 separate experiments.

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