Efficient production of Fah-null heterozygote pigs by chimeric adeno-associated virus-mediated gene knockout and somatic cell nuclear transfer
✍ Scribed by Raymond D. Hickey; Joseph B. Lillegard; James E. Fisher; Travis J. McKenzie; Sean E. Hofherr; Milton J. Finegold; Scott L. Nyberg; Markus Grompe
- Publisher
- John Wiley and Sons
- Year
- 2011
- Tongue
- English
- Weight
- 466 KB
- Volume
- 54
- Category
- Article
- ISSN
- 0270-9139
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✦ Synopsis
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.