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Figure 1. Development of the control FIV(
'lac) vector with inactive ß-galactosidase gene. (A) The reporter gene lacZ was inactivated after deletion of a critical placZ DNA fragment containing the ß-galactosidase gene transcription initiation site by restriction enzyme-mediated excision and re-ligation of the backbone vector. (B) The structure of mutated FIV('lac) and wild-type FIV(lacZ) viral vectors was confirmed by PCR following transient transfection into the murine cell line NIH 3T3. The presence of viral DNA in cells was detected by a 444-bp DNA band utilizing the "FIV" primers (as depicted in panel A). The complete structure of the lacZ gene was confirmed by a 1.7-kb DNA band utilizing the lacZ primers (depicted as UP, LP in panel A). In the case of the mutated FIV('lac), there was a lack of the 1.7-kb DNA band as the annealing site for the lower primer LP was excised. (C) Deletion of the lacZ transcription initiation sequence in the FIV('lac) resulted in inactivation of the ß-galactosidase reporter gene, as demonstrated by the lack of X-gal staining compared with (D) the FIV(lacZ) vector.
