Targeting strategy and molecular verification of myoglobin disruption.

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Targeting strategy and molecular verification of myoglobin disruption. Targeting strategy and molecular verification of myoglobin disruption. (a) Structures of the WT and mutated alleles (mut) and the targeting vector are shown. (Restriction sites: B, BamHI; Bg, BglII; HIII, HindIII; HcII, HincII; Xb, XbaI. neoR, neomycin resistance gene). (b) Southern blot analysis of BglII-digested DNA from WT (+/+), heterozygous (+/−), and homozygously mutated (−/−) mice. The BamHI-HindII-fragment indicated in a was used as a probe. Hybridizing fragments of 2.9 kb (WT allele) and 12 kb (mutated allele) were detected. (c) Northern blot analysis of cardiac RNA isolated from the three genotypes. (d) SDS/PAGE analysis of protein patterns of myo−/− and WT hearts. Two-hundred micrograms of cardiac proteins were separated on a 12.5% SDS gel and were stained with Coomassie brilliant blue. Five micrograms of horse myoglobin were loaded as control. (e) Morphology of myoglobin-deficient hearts. WT (left) and myo−/− hearts (right) were perfused free of blood and were photographed. Axel Gödecke et al. PNAS 1999;96:18:10495-10500 ©1999 by National Academy of Sciences