KCNJ11 knockout morula re-engineered by stem cell diploid aggregation

KCNJ11-encoded Kir6.2 assembles with ATP-binding cassette sulphonylurea receptors to generate ATP-sensitive K⁺ (K_ATP) channel complexes. Expressed in tissues with dynamic metabolic flux, these evolutionarily conserved yet structurally and functionally unique heteromultimers serve as high-fidelity rheostats that adjust membrane potential-dependent cell functions to match energetic demand. Genetic defects in channel subunits disrupt the cellular homeostatic response to environmental stress, compromising organ tolerance in the adult. As maladaptation characterizes malignant K_ATP channelopathies, establishment of platforms to examine progression of K_ATP channel-dependent adaptive behaviour is warranted. Chimeras provide a powerful tool to assay the contribution of genetic variance to stress intolerance during prenatal or post-natal development. Here, KCNJ11 K_ATP channel gene knockout ↔ wild-type chimeras were engineered through diploid aggregation. Integration of wild-type embryonic stem cells into zona pellucida-denuded morula derived from knockout embryos achieved varying degrees of incorporation of stress-tolerant tissue within the K_ATP channel-deficient background. Despite the stress-vulnerable phenotype of the knockout, ex vivo derived mosaic blastocysts tolerated intrauterine transfer and implantation, followed by full-term embryonic development in pseudopregnant surrogates to produce live chimeric offspring. The development of adult chimerism from the knockout ↔ wild-type mosaic embryo offers thereby a new paradigm to probe the ecogenetic control of the K_ATP channel-dependent stress response.