Southern blot was conducted using an Fah-specific probe located outside of the homologous targeting region (Fig. 2C). The

targeting frequencies obtained using the rAAV-DJ were extremely high, with an average targeting frequency of 5.4% (range, 2.29%-8.50%) (Table 2). Confirmed Fah-null heterozygote fibroblasts that had been in culture https://www.selleckchem.com/products/MLN8237.html for 15-19 days were frozen down for SCNT. Southern blot using a neo-specific probe was used to identify clones with targeted Fah alleles that were free of other random integration events (data not shown). All double-positive PCR clones were also positive by Southern blot and no additional random integration events or sequence anomalies were observed. To produce heterozygote pigs, Fah-null-targeted fetal fibroblasts were used as nuclear donors for transfer to enucleated oocytes. Then to each of four surrogate females, 134 embryos were transferred, with only one surrogate reaching full

term and delivering five viable offspring by natural vaginal birth. PCR and Southern blot revealed that all five of the offspring were Fah-null heterozygotes (Fig. 3). One of the newborn Fah-null heterozygote piglets was euthanized 24 hours after birth because of failure to thrive. Figure 4 shows a picture of the surviving four Fah-null heterozygote piglets hours after their birth. Upon reaching reproductive maturity, female Fah-null heterozygote pigs were bred to male wildtype pigs. The Fah knockout allele was inherited by newborn NADPH-cytochrome-c2 reductase piglets with the expected Mendelian result: 50% of males and 55% of females carried the knockout allele (data not shown). GPCR Compound Library research buy Fah-null heterozygotes were then compared to wildtype littermates.

Fah-null heterozygote piglets were phenotypically normal and had normal levels of the amino acids phenylalanine and tyrosine, as well as the tyrosinemia type 1 marker succinylacetone, which indicated normal tyrosine metabolism in these animals compared to wildtype sibling controls (Table 3). In addition, hematoxylin and eosin (H&E) staining of livers of Fah-null heterozygotes appeared histologically normal and were positive for FAH by immunostaining within the hepatocytes (Fig. 5A,B). However, quantitative PCR (qPCR) analysis revealed a 55% reduction of the Fah transcript, in addition to a reduction of the overall FAH protein seen by western blot analysis from livers of Fah-null heterozygotes when compared to wildtype animals (Fig. 5C,D). Finally, FAH enzyme activity can be measured by fluorometric quantification. FAH converts 4-fumarlacetoacetate (FAA) to acetoacetate and fumerate. The loss of FAA is detected as decreased absorbance at 330 nm. In accordance with FAH protein levels, the Fah-null heterozygotes showed reduced FAH enzyme activity when compared to their wildtype littermates (Fig. 5E).