Figure 9. CR1-PCR analysis for genomic integration of YFP in chicken cells, transduced with FIV-YFP vectors.

Schematic representation of the two-step PCR approach using the CR1- and LTR-specific primers. Diluted (1∶1000) mixes from the first long PCRs, employing CR1 and LTR primers, were used as templates for short nested PCRs with nested LTR primers. In the absence of genomic integration, no signals are expected in the nested PCR. This is indicated by the control long PCR with no CR1 primers. B. Nested PCR products separated on 2% agarose gel, obtained using as templates the first long-PCR DNAs, which were prepared 14 days after in vitro transduction with FIV-YFP, from E11 liver and muscle cells. The primers used for the first long PCR are indicated: the LTR primers were 5′LTR (5′L) and 3′LTR (3′L); the CR1 primers were CR1-1F (C1F), CR1-1R (C1R), CR1-3F (C3F) and CR1-3R (C3R). The nested primers are indicated in Materials and Methods. Neither the controls without CR1 primers, nor the control using DNA from non-transduced cell cultures gave a signal. The expected size fragments were 120 bp for the 5′LTR and 110 bp for the 3′LTR. The products were confirmed by sequencing. These results indicate genomic integration of FIV-YFP-derived cDNA in the host chicken cells. M, molecular weight markers.