Construction of Dkk1 Ads. Dkk1 cDNA was amplified from E17.5 mouse embryo cDNA by PCR, using the forward primer 5'-GAT CGG GGC CCA GCC GGC CAC CTT GAA CTC AGT TCT CAT CAA T-3' and the reverse primer 5'-GAT CGG ATC CTC AAT GGT GAT GGT GAT GAT GCT TGT CAT CGT CGT CCT TGT AGT CGT GTC TCT GGC AGG TGT GGA GCC T-3', which incorporated C-terminal FLAG and His₆ epitope tags. The PCR product was cloned into pCR2.1 (Invitrogen), was sequenced and was subcloned SfiI–SalI as an in-frame fusion with the IgK signal peptide downstream of the human CMV promoter of the Ad shuttle plasmid, Add2 SecTag, a variant of Add2 (1). For murine Dkk1-HA containing an N-terminal HA and C-terminal FLAG and His₆ epitope tags, the Dkk1A insert was excised SfiI–SalI and ligated in-frame into SfiI–SalI–cut Ad shuttle plasmid Add2 Display, a variant of Add2 (1) containing a 5' IgK signal peptide and an HA tag. The Dkk1 and Dkk1-HA inserts were cloned into the E1 region of E1^-E3^- Ad strain 5 as using homologous recombination, followed by Ad production in 293 cells and CsCl gradient purification of virus as described (1, 2). The negative control virus Ad Fc expressing a murine IgG2a Fc fragment has been described (1).

b -catenin Stabilization Assay. L cells were grown in DMEM containing 10% FBS and seeded in 24-well plates at a density of 2 × 10⁵ cells per well. The cells were treated with 125 ng/ml Dkk1 purified over Ni-agarose from adenoviral supernatant for 2 h, after which purified Wnt3a protein (Nusse Laboratory, Stanford, CA) was added for an additional 3 h (1:8,000). Cells were washed in PBS and lysed in TNT buffer (150 mM NaCl/50 mM Tris·HCl, pH 7.5/1% Triton X-100). The cell lysates were analyzed for b -catenin levels by using Western blotting and anti-b -catenin mAb (BD Transduction Laboratories, Stanford, CA).

Luciferase Reporter Assays. The 293T cells were seeded in 24-well plates at a density of 1 × 10⁵ cells per well. Plasmids transfected are as follows (m g per well): pTOPFLASH, 0.1; EF-LacZ, 0.1; PGKWnt3a, 0.3; Add Dkk1, 0.3. Total DNA transfected was normalized to 0.8 m g per well by using PGK vector. Luciferase assays were performed using the Dual-Light reporter gene assay system (Tropix, Bedford, MA). Luciferase activity was normalized against b -galactosidase activity and all assays were performed in triplicate.

Quantitation of Proliferative Index. Ki67-positive epithelial cells were quantitated on 3-5 high-powered fields for each portion of the gastrointestinal tract. Fields were selected for similar tissue planes and an equivalent number of anatomic structures (e.g., villi) were analyzed on each field. The observer was blinded to the treatment conditions of the mice.

1. Kuo, C. J., Farnebo, F., Yu, E. Y., Christofferson, R., Swearingen, R. A., Carter, R., von Recum, H. A., Yuan, J., Kamihara, J., Flynn, E., et al. (2001) Proc. Natl. Acad. Sci. USA 98, 4605-4610.

2. Chartier, C., Degryse, E., Gantzer, M., Dieterle, A., Pavirani, A. & Mehtali, M. (1996) J. Virol. 70, 4805-4810.