PCR analysis of GCV-sensitive and GCV-resistant cells. The PCR reaction detects LTR sequences of the retroviral vector.²⁰  Tumors with a negative Southern blot (TK probe; data not shown)²³  and a positive PCR reaction were numbers 2-1, 2-5, 3-4, and 5-2. Clones with ambiguous results were repeatedly analyzed. Starting material for PCR was gDNA; M, 100-bp ladder DNA marker; EL-4, untransduced EL-4 cell gDNA; P1 and P2, primers LTR1 and LTR2 tested on number 1 gDNA; g, a split from isolated tumor cells had been cultivated further and was selected additionally with G418 before gDNA extraction; mc-1 to mc-5, tumor cells isolated from relapsed mice that originally received transplants of vector-transduced mass culture (mc) cells; e, tumor cells had been isolated from mice that were treated with GCV beginning one day after tumor cell inoculation; vt, EL-4 cells isolated after GCV selection in vitro; and n, tumor cells isolated from mice that had not been treated with GCV. DNA from clones with a negative result in the LTR-PCR was successfully amplified using primers annealing to the endogenous Xist gene (see “PCR” in “Materials and methods”).