AAV-DJ is an artificial chimeric AAV vector containing hybrid capsid sequences from three naturally occurring serotypes (AAV2, 8, and 9). The AAV-DJ vector was used to deliver the knockout construct

Ivacaftor in vivo to fetal pig fibroblasts with an average knockout targeting frequency of 5.4%. Targeted Fah-null heterozygote fibroblasts were used as nuclear donors for somatic cell nuclear transfer (SCNT) to porcine oocytes and multiple viable Fah-null heterozygote pigs were generated. Fah-null heterozygotes were phenotypically normal, but had decreased Fah transcriptional and enzymatic activity compared to wildtype animals. Conclusion: This study is the first to use a recombinant chimeric AAV vector to knockout a gene in porcine fibroblasts for the purpose of SCNT. In using the AAV-DJ vector we observed targeting frequencies that were higher than previously reported with other naturally occurring serotypes.

We expect that the subsequent generation of FAH-null homozygote pigs will serve as Y-27632 solubility dmso a significant advancement for translational research in the areas of metabolic liver disease, cirrhosis, and HCC. (HEPATOLOGY 2011;) In humans, hereditary tyrosinemia type I (HT1) is a severe, autosomal recessive inborn error of metabolism caused by deficiency of fumarylacetoacetate hydrolase (FAH), a metabolic enzyme that catalyzes the last step of tyrosine metabolism.1 Affected children develop micronodular cirrhosis and 28% of children older than 2 years develop hepatocellular carcinoma (HCC) if untreated.2 We have previously

created a small animal model of HT1 by generating Fah mutant mice by gene targeting in embryonic stem cells.3 The phenotype of these mice is analogous to many of the key features of the human disorder, including the formation of liver cancer, and has proven to be an DNA Damage inhibitor important research model for both HT1 and HCC.4-7 However, this mouse model fails to recapitulate all of the aspects of the human disorder, most importantly cirrhosis. Similar deficits in small-animal models of other human disease have been reported as well. In cystic fibrosis (CF), multiple mice and rabbit models were engineered to contain several of the common genetic mutations seen in humans. However, these models failed to fully reproduce the disease phenotype observed in humans.8, 9 The pig is an appropriate research model because of its similarity in size, anatomy, and biology to the human.10 This led researchers to create a porcine model of CF by using adeno-associated virus (AAV)-mediated gene targeting in combination with somatic cell nuclear transfer (SCNT) to create their pig model of CF, which has now been shown to display the characteristic manifestations of CF seen in humans.11, 12 The CF pig was the first time AAV had been used to generate a porcine gene-knockout model. Up to this point, the generation of large-animal models of disease had been hindered by the inability to apply mouse embryonic stem cell targeting approaches to nonmurine models.