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Background: Recent research at Duke with collaborators has shown that de novo mutations in ATP1A3 gene cause alternating hemiplegia of childhood (AHC; Heinzen et al, 2012). Missense mutation in ATPA13- D801N is the most frequently seen in AHC patients. Goals: Create and characterize a mouse model carrying the most common mutation causing AHC in humans. Methods: ES cell approach followed by homologous recombination, microinjection in blastocysts and breading of chimeric mice with wild type to generate the knock in mice. Results: A small plasmid containing of our gene of interest, with the desired mutation, was removed from bacterial artificial chromosome and linearized construct introduced into ES cells (129SvEv) by electroporation resulting homologous recombination at the shared sequence in one allele, during which two crossover events replaced the wild type gene with the targeting construct. Using genomic southern blot the desired mutation in the ES cells DNA was confirmed. Heterozygous ES cells were expanded, microinjected into blastocysts harvested from C57BL/6 mice, and implanted into uterus of pseudopregnant females. Male chimeras were mated with C57BL/6 females. PCR genotyping confirmed heterozygote agouti mice which were further mated with Flp mice to remove antibiotic(neo) cassette. The resultant litters were subsequently mated to make a colony for behavioral and neurophysiological studies. Conclusions: We have developed knock-in mouse model that has the most common mutation affecting humans with AHC.

AHC; ATP1A3; D801N; E815K; Knock in mouse