Activating a patient-relevant mutation of a genetically defined heart disease in a humanized pig model

Complex diseases such as progressive cardiomyopathies are often insufficiently reflected in small animal and in vitro models. Large animal species such as pigs provide a valuable alternative, but constitutive genetic manipulation (cGM) has not yet been applied to pig in an effective manner, mainly due to biological and logistic limitations. Here, we describe the generation of a novel humanized model for PLN-mediated cardiomyopathy and compare different methods of activating a pathogenic R14del mutation by Cre-mediated recombination. Both, the Cre- treatment of pig primary cells before somatic cell nuclear transfer as well as the micro-injection of Cre-encoding mRNA into zygotes, were similarly efficient in delivering piglets with an activated R14del mutation. Alternatively, administration of Cre-encoding AAV into piglets was sufficient in cGM, albeit at to a varying extent. Together, we here describe a highly effective process to establish complex cGM traits in pig and demonstrate that the lack of Cre-driver lines can be compensated by various interventations during reproduction or post-natally.