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Caption Generation of Ireb2tm1.1Eal/Ireb2tm1.1Eal mice. (A1) Schematic representation of exons I-VII of the murine Ireb2 (Irp2) gene. Exons (black boxes) and introns (black lines). (A2) Schematic diagram of the targeting vector. A genomic clone (p11.1) containing exons III-VII encoding amino acids 37-233 in Ireb2 was isolated from a mouse 129 genomic library. A ScaI-PstI fragment from p11.1 (dotted lines) was subcloned into Bluescript SK to generate the targeting vector. A self-excision cassette containing neomycin (Neor) linked to Cre-recombinase (Cre) was inserted into exon III of the mouse gene. This cassette (pACN) contains the Cre gene (driven by the testes-specific angiotensin-converting enzyme (tACE promoter)) linked to the Neor gene (driven by the polymerase II promoter), and is flanked by loxP sites allowing for excision of Neor as it passes through the male germ line. This cassette also contains human hIrp1-Flag sequence fused in-frame to exon III of Ireb2 for experiments related to functional replacement of Ireb2 with Irp1. For reasons discussed below, Irp1-Flag was not detected in any tissues examined. Thymidine kinase (Tk1 and Tk2) genes were inserted at the 5' and 3' ends of the targeting vector to select against random insertion. (A3) Predicted structure of the Ireb2 replacement allele after homologous recombination and germ-line induced self-excision. The targeting vector was electroporated into R1 ES cell line and heterozygous cells in which a homologous recombination event occurred were identified by PCR and Southern blot analysis of ScaI-digested DNA probed with 3' EXT and Neo probes. A targeted ES cell clone (F4) was microinjected into C57BL/6J blastocysts to generate chimeric animals. A chimera was bred to C57BL/6J mice and resulting chimeric progeny was identified by coat color and for the presence of the allele by PCR and Southern blotting.
Copyright This image is from Zumbrennen-Bullough KB, PLoS One 2014;9(6):e98072, and is displayed under the terms of the Creative Commons Attribution 4.0 International License. J:217335
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Ireb2tm1.1Eal iron responsive element binding protein 2; targeted mutation 1.1, Elizabeth A Leibold

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