Optimized concatemeric array transfection for efficient stable producer cell engineering. (A) Diagrammatic representation of vector (long arrows) and ble (short arrows) ligation events, with the theoretically expected molar frequency of that event in the reaction and the fragment sizes after EcoRI digestion. E, location of EcoRI sites. Solid interior rectangles indicate approximate location of probe for Southern blot in panel B. Note that the 3.7-kb TT_v-v fragment does not contain a probe target and so is not visualized on the Southern blot. HH, head-to-head; TT, tail-to-tail; HT, head-to-tail; v-v, vector-to-vector ligation; b-v, ble-to-vector ligation. (B) Southern blot analysis of EcoRI-digested, concatemerized vector DNA after selection in GPRG packaging cells for the indicated number of days, probed with a fragment of the vector genome (indicated in panel A). U, untransfected GPRG cells. Sizes from standards are indicated (in kb) at left. Designations in the center correspond to those in panel A. Real-time PCR quantitated vector copy numbers for each population are indicated at the bottom (in copies/cell).