FIG. 1.

MLV-based RCR vector constructs and experimental setup. (A) Mo-MLV-based (white vector backbone) and Akv-MLV-based (light gray vector backbone) vectors contain an IRES-eGFP cassette inserted either immediately adjacent to the 3′ end of the env gene or in the 3′-LTR U3 region. A glutamine-to-arginine transition at codon position 110 of the gag gene of vector AkvB4070A-eGFP is denoted by an asterisk. (B) Experimental setup for testing the genomic stability of vectors. Infection cycle 1 was initiated by inoculating cells with an MOI of 0.001, passaging cells 2 days later, and subjecting them to FACS analysis, followed by passaging and FACS analysis every 2 or 3 days until the percentage of eGFP-expressing cells no longer increased from one passage to the next. Four days postinfection, fresh cells were inoculated with three different dilutions of cell-free virus-containing supernatant to initiate infection cycle 2. Cells infected with a dilution factor resulting in infection levels of between 1% and 10% in FACS analyses 2 days postinfection were chosen for further analysis. This process was repeated for multiple serial infection cycles.