Efficient production of Fah-null heterozygote pigs by chimeric adeno-associated virus-mediated gene knockout and somatic cell nuclear transfer

Raymond D. Hickey, Joseph B. Lillegard, James E. Fisher, Travis J. McKenzie, Sean E. Hofherr, Milton J. Finegold, Scott L. Nyberg, Markus Grompe

Research output: Contribution to journalArticle

51 Scopus citations

Abstract

Hereditary tyrosinemia type I (HT1) results in hepatic failure, cirrhosis, and hepatocellular carcinoma (HCC) early in childhood and is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase (FAH). In a novel approach we used the chimeric adeno-associated virus DJ serotype (AAV-DJ) and homologous recombination to target and disrupt the porcine Fah gene. 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 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 a significant advancement for translational research in the areas of metabolic liver disease, cirrhosis, and HCC.

Original languageEnglish (US)
Pages (from-to)1351-1359
Number of pages9
JournalHepatology
Volume54
Issue number4
DOIs
StatePublished - Oct 1 2011

ASJC Scopus subject areas

  • Hepatology

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