AAV vectors have been shown to be able to introduce specific mutations, including insertions,

into homologous chromosomal sequences of many cell types and species.13, 14 In addition, AAV-mediated gene targeting has been shown to be more efficient than conventional techniques based on transfection or electroporation of plasmid constructs.15-17 The goal of this study was to develop a more efficient method to create pig knockout models of human diseases and to create Fah-null heterozygote pigs to AUY-922 nmr be used to generate homozygote Fah-null animals for future studies related to metabolic liver disease, cirrhosis, HCC, and cell and gene therapy. We used for the first time the novel chimeric AAV-DJ serotype to disrupt the porcine Fah gene by targeted gene knockout by homologous recombination. We report here on the successful and efficient generation of targeted Fah-null heterozygote fibroblasts and their use by SCNT to generate Fah-null heterozygote pigs. AAV, adeno-associated virus; CF, cystic fibrosis; FAH, fumarylacetoacetate hydrolase; HCC, hepatocellular carcinoma; HT1, hereditary type I tyrosinemia; http://www.selleckchem.com/products/epz-6438.html SCNT, somatic cell nuclear transfer. Genomic DNA was isolated and purified (Qiamp; Qiagen) from pig fetal liver. Two fragments of DNA, adjacent to exon 5 of the Fah locus of chromosome 7, were amplified

using primers MG2616 and MG2678 (left homologous recombination arm; 1479 basepairs [bp]) and MG2619 and MG2680 (right homologous recombination arm; 1523 bp) and a high-fidelity polymerase (Phusion; Finnzymes). Primers were designed based on the domestic pig working draft genomic sequence (GenBank accession numbers CU468492 and CU467891).

These polymerase chain reaction (PCR) products were subcloned into pCR-Blunt II-TOPO (Invitrogen) and confirmed by restriction digest and sequencing. The overall strategy to knockout the Fah gene was to insert an in-frame stop codon and a neomycin-resistance cassette (PGK-neo) into exon 5 of the porcine Fah gene. To generate a PGK-neo expression cassette, with an additional in-frame 上海皓元医药股份有限公司 TGA stop codon at the 5′ end, a 1681 bp fragment was amplified using primers MG2622 and MG2679, subcloned into pCR-Blunt II-TOPO, and confirmed by restriction digest and sequencing. To generate the complete targeting vector, each fragment was sequentially subcloned into pcDNA3.1- (Invitrogen) and orientation confirmed by restriction digest and sequencing. Once the full-sized 4683 bp-targeting construct was generated, it was cloned into an AAV2 plasmid backbone, thus providing it with the inverted terminal repeat (ITR) sequences required for viral packaging. Plasmids containing the AAV-DJ shuffle capsid sequences were generously provided by Dr. Mark Kay at Stanford. AAV-DJ virus containing the Fah-null construct was produced using a standard triple plasmid transfection protocol, as described.