Costello Syndrome (CS) is a rare, complex, developmental disorder characterized by a number of features including— failure to thrive, characteristic facies, delay in intellectual development, hypertrophic cardiomyopathy, arrhythmia, and predisposition to both benign and malignant tumors. Past studies identifying gain-of-function mutations in the HRAS gene as the basis of human CS, and strong conservation with the mouse ortholog Hras1, have led to the development of a Gly12Val (G12V) mouse model of CS. To extend animal modeling studies to include various allelic forms of the disease, we are developing five additional mouse models of CS. Using a recombineering-based “knock-in” approach, we have completed construction of five vectors, each containing a loxP-flanked, neomycin resistance (Neor) selection cassette in intron 1 of Hras1 and site-directed mutations encoding each of the following five pathogenic alleles — G12A encoded by GCA, G12A encoded by GCC, G12S encoded by AGC, G12V encoded by GTA and G12V encoded by GTT. Four of the five strains have been completed creating animal models for the G12AGCA, G12AGCC, G12VGTA and G12VGTT forms of the disease. Suppression of each CS-causative allele by the oppositely transcribed Neor transcript can be relieved by breeding affected mice to inner cell mass- or germline-specific “deleter” Cre lines such as Hprt-Cre or Alpl-Cre, respectively. Abnormalities have been observed for all mutant alleles but appear to be most pronounced in the two G12V lines where there is a higher degree of lethality than in the two G12A alleles. For all alleles, mutant mice display a tremor and appear less robust than their wild type littermates. Craniofacial abnormalities are also apparent in all mutant lines. Preliminary phenotyping of a small number of mutants suggests cardiac defects (including mitral valve abnormalities and abnormal ECG patterns), enlarged regions of the brain (including the pineal gland), possible neoplasia and skin abnormalities. Each strain will be deposited into The Jackson Laboratory Repository. Together, these five models hold the potential for uncovering allelic and codon preference influences on development and neoplasia in CS, identifying CS modifying genes, and dissecting tissue-specific facets of CS using spatially-controlled Cre driver lines.