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. 2017 Oct 9;45(22):12766–12779. doi: 10.1093/nar/gkx896

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Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Figure 3. — Restoring monoallelic expression of Igf2 in LOI cells rescues their differentiation defects. (A) Genetic scheme to restore monoallelic expression of Igf2 in LOI cells. In mouse genetic nomenclature, maternal alleles are indicated first. For example, ΔICR/Igf2- means that the maternal chromosome carries the ICR deletion and the paternal chromosome carries the Igf2 deletion described in the text. Here we crossed ΔICR/ΔICR mice with Igf2+/- mice to obtain two genotypes of interest: ΔICR/Igf2+, which phenocopies ΔICR/ΔICR (extra Igf2, low H19) and ΔICR/Igf2- which genetically rescues Igf2 expression while maintaining LOI levels of H19. (B, C) Ablation of the paternal Igf2 allele in LOI cells restores expression of IGF2 to wild type levels. (B) Immunoblot analyses. (C) ELISA analyses. (D) H19 expression relative to GAPDH was determined by qRT-PCR. Expression in wild type cells is set at 1. The ΔICR/Igf2- and ΔICR/Igf2+ cells have significantly downregulated H19 levels, similar to ΔICR/ΔICR. (E, F) Ablation of paternal Igf2 in LOI cells rescues differentiation defects. ΔICR/Igf2- myotubes have improved morphology with near-normal fusion, while ΔICR/Igf2+ cells have aberrant myotube morphology similar to LOI cells. (E) DAPI (blue) and Myh3 (green) staining. (F) Quantitation of cell fusion. In all experiments, cells were grown for 72 h in differentiation media and at least 3 independent cultures were analyzed. Statistical significance was evaluated by t-test, comparing to wild type controls. **P < 0.01; ***P < 0.001.