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. 2000 Apr 11;97(9):4802–4807. doi: 10.1073/pnas.090065597

Figure 1.

Figure 1

Construction, molecular analysis, and transgene expression of ADV.mEnd. (A) The expression cassette consisting of human elongation factor-1 α (EF-1α) promoter, the rat insulin leader sequence, the cDNA of murine endostatin, and the bovine growth hormone poly(A) (bGHPA) was cloned in an adenovirus shuttle vector. Recombinant adenovirus was generated on cotransfection of the shuttle plasmid with the adenovirus backbone plasmid pJM-17. (B) Rearrangement of HindIII restriction fragments (loss of a 3,437-bp fragment (arrow), emergence of a 1,003-bp fragment (data not shown), and other minor band shifts of the adenovirus backbone plasmid (lane 1) as compared with the recombinant ADV.mEnd (lane 2) confirmed the correct insertion of the transgene into the E1 region in the adenovirus. (C) Ten percent reducing SDS/PAGE of serum-free concentrated supernatant of virally transduced breast cancer cells. Retention on 50-kDa molecular mass cut-off column, ADV.mEnd (lane 1) and ADV.β-Gal (lane 2); flow-through of 30-kDa cut-off column, ADV.mEnd (lane 3) and ADV.β-Gal (lane 4); retention on 30-kDa molecular mass cut-off column, ADV.mEnd (lane 5) and ADV.β-Gal (lane 6). (D) Western blot of the same supernatant as shown in C. Lanes 1 and 2 are the same as in C, and lanes 3 and 4 correspond to lanes 5 and 6 in C. M, molecular mass markers.