Retroviral transduction of CV-1 cells. (A) Retroviral constructs. Each Wnt cDNA was digested with restriction enzymes to eliminate 5′ and 3′ UTR and subcloned into the MSCVneoEB vector at the EcoRI and Xho I sites downstream of the LTR promoter. A line represents the 5′ and 3′ UTRs and an open box represents the coding sequence. Numbering for each cDNA begins at the first nucleotide of the initiation codon. (B) RT-PCR analysis of transduced CV-1 cells. Specific primers for each of the Wntgenes were used to analyze expression of the transduced genes in cDNA (+RT) from CV-1 cells transduced with the MSCVneoEB vector (CV1), from each transduced population (CV1/5A, CV1/2B, CV1/10B), and from a no-template control (−). A control lacking RT in the cDNA synthesis step (−RT) confirms that the PCR bands in the +RT samples are specific for RNA. PCR with GAPD primers was also performed (GAPD) to confirm the ability of the isolated RNA to be PCR amplified and to standardize for loading.