Adeno-associated virus-mediated TNF-α gene targeting in HeLa cells. (a) Genomic fragment containing the human TNF-α gene (top) and the recombinant adeno-associated virus (rAAV) TNF-α targeting vector (bottom). Arrows mark nested primers used for PCR screening of targeting events. Restriction sites and probes used for Southern blot confirmation of targeting events are also shown. The left and right homologous arms (Homologous Arm) in the AAV targeting vector contain endogenous sequence from the wild-type TNF-α allele. (b) Southern blot analysis of cells derived from the targeted intermediate clone Tg#28zeoR, using restriction sites and the TNF-α probe indicated in panel (a). Parental HeLa cell genomic DNA is shown in lanes 1, 3 and 5 and targeted clone Tg#28zeoR genomic DNA in lanes 2, 4 and 6. (c) Strategy used to generate the final TNF-α targeted R-Luc reporter cell line by LoxP/Cre-mediated excision of the Zeocin selection cassette. (d) Southern blot analysis of the zeocin-resistant targeted intermediate clone (Tg#28zeoR, marked as Z+), and the Zeocin-sensitive targeted cell line (Tg#28zeo-, marked as Z-). Arrows to the left of the blots indicate the DNA fragments cut from non-targeted (solid) and gene-targeted (open) TNF-α alleles. (e) Renilla luciferase (R-Luc) reporter activity in the TNF-α targeted intermediate clone Tg#28zeoR, the Tg-Zeo- cell pool following excision of the selection marker, and from 5 individual Tg-Zeo- clonal cell lines (#1–5) isolated from the Tg-Zeo- cell pool. Results represent the mean (+/-SEM, N = 4). One-way ANOVA demonstrated no significant difference (p > 0.05) between the targeted cell pool and the five individual targeted clonal cell lines isolated from the pool.