The LasBD retroviral vector. (A) Retroviral vector LasBD containing the tyr22-DHFR gene transcriptionally regulated by an internal β-actin promoter and two 20-mer anti-b3a2 AS modified sequences regulated by the MoMuLV-LTR. The retroviral vector, LBD, contains the tyr22-DHFR gene transcriptionally regulated by an internal β-actin promoter but no anti-BCR/ABL antisense sequences. (B) Northern blot analysis showing equal expression of the LTR-regulated AS-DHFR message (2.6 kb) and the internal β-actin promoter-regulated DHFR message (1.3 kb). Expression of both mRNAs is depicted for PA317 packaging cells, LasBD-32D^p210 cells recovered after ex vivo selection in MTX and IL-3, and LasBD-32D^p210 cells recovered 70 days after in vivo infusion of bulk-selected LasBD-32D^p210 cells into syngeneic C3H mice. (C) Secondary structure of the anti-b3a2 AS sequence, determined based on a dynamic program algorithm of the minimum free energy conformation of the RNA using the Program Manual for the Wisconsin Package (version 8, September 1994; Genetics Computer Group, Madison, WI).41 42 The optimal stable conformation of the modified double antisense mRNA indicates that 5 to 10 bases surrounding the b3a2 breakpoint are single-stranded.