title: Efficient production of Fah-null heterozygote pigs by chimeric adeno-associated virus-mediated gene knockout and somatic cell nuclear transfer.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Papé Family Pediatric Research Institute & Oregon Stem Cell Center, Oregon Health & Sciences University, Portland, OR 97239, USA. hickeyr@ohsu.edu
authors.forename: Raymond D
authors.initials: RD
authors.lastname: Hickey
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Lillegard
authors.forename: James E
authors.initials: JE
authors.lastname: Fisher
authors.forename: Travis J
authors.initials: TJ
authors.lastname: McKenzie
authors.forename: Sean E
authors.initials: SE
authors.lastname: Hofherr
authors.forename: Milton J
authors.initials: MJ
authors.lastname: Finegold
authors.forename: Scott L
authors.initials: SL
authors.lastname: Nyberg
authors.forename: Markus
authors.initials: M
authors.lastname: Grompe
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015139:Blotting, Southern; D002678:Chimera; D000229:Dependovirus / Q000235:genetics*; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors; D006579:Heterozygote; D059765:Homologous Recombination / Q000235:genetics / Q000502:physiology; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism*; D023421:Models, Animal; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013552:Swine / Q000235:genetics*; D020176:Tyrosinemias / Q000235:genetics / Q000503:physiopathology
chemical_list: D006867:Hydrolases; C020156:fumarylacetoacetase