Figure 4

Distribution and sequence analysis of vector recombinants. (A) Schematic representation of vector mutants detected by PCR using primers annealing in the MLV 4070A env and eGFP gene indicating the distribution of the deleted sequences. Numbers indicate distances of vector border regions from the 5' end of the IRES. Annealing positions of sequencing primers are indicated by black arrows. Coloured horizontal lines indicate the positions of single deleted sequences in mutants isolated from in vitro infected cell lines (NIH-3T3 (blue), HEK293 (red), U87-MG (green)) and from in vivo infected U87-MG tumours (black). (B) Sequence analysis of deletion junctions. Nomenclature of the PCR products is according to Fig. 3. Intact (capital letters) and deleted sequences (lower case letters) are separated by vertical text indicating the regions where the deletions occurred. Bold numbers display the distances of the 5' and 3' deletion junctions from the 5' end of the IRES, which is set as 1. Lengths (nt) of deleted regions are indicated by a delta symbol. Repeats flanking the deleted sequences are indicated by underlined letters (continuous lines represent direct repeats, dotted lines represent inverted repeats). a* = insertion of 81 nt representing the 5' part of the 3' LTR U3 region, translocated from the 5' end of the transgene cassette and reconstituting the U3 region, b** = recurring 75 nt deletion occurring in the U3 region, detected in recombinants AC2-1, AC2-2, AU2-1, AU2-3, AU2-4, and AUN-1, c*** = 90 nt deletion found in mutant vectors ACN-2 and AU2-4.