FIG. 2.

Analysis of homologous recombination of HC-Ad vector DNA with chromosomal DNA in vivo. (A) Schematic of the HC-AdSLS14 vector carrying a 12.3-kb genomic fragment of the murine DNA and the scheme of homologous recombination (X) whereby exon 5 in the FAH locus may be replaced by the targeting vector. ITR, Ad5 inverted terminal repeat; Ψ, Ad5 packaging signal; murine FAH fragment, 12.3-kb murine FAH genomic DNA fragment beginning at nt 91754097 of the murine FAH gene from introns 1 to 9; E2 to E9, wild-type exons 2 to 9, respectively; E5*, mutated exon 5 with NeoR insertion; Neo, NeoR gene; stuffer DNA, 16-kb stuffer from human HPRT genomic DNA (nt 1799 to 17853 of human HPRT gene). The positions of the primers used in the PCR to demonstrate the presence of an uninterrupted exon 5 in the livers are indicated as the horizontal arrows FAHA, FAHB, and FAHC. (B) The 2% agarose gel used for separation of the PCR products; Lanes: −, water controls; f+* and f+, C57/BL6 and 129 mouse controls; respectively; p, pSLS14 plasmid control; f−, FAH^−/− mouse negative control; 10, FAH^−/− mouse injected with the first-generation Ad vector negative control; 9, transplantation recipient of HC-AdSLS16 vector negative control; 4 to 8, transplantation recipients; 1 to 3, injected animals. (C and D) Representative photomicrographs of liver sections stained with hematoxylin-eosin. FAH expression is detected with a polyclonal rabbit antibody to rat FAH.