Immunomagnetic selection and Southern blot analysis.

(A) Immunomagnetic selection of Fas-engineered T lymphocytes. Fas-transduced primary human T lymphocytes were selected with anti-LNGFR–coated magnetic beads, as described in “Materials and methods.” Before selection, cells were 10% to 50% LNGFR-positive; after selection, they were routinely more than 95% to 99% pure. Histogram plots show the LNGFR staining profile of transduced (filled histograms) and untransduced cells (open histograms). (B) Southern blot analysis of Fas-transduced T lymphocytes. Immunomagnetically sorted, Fas-transduced primary human T lymphocytes were either challenged with 10 nM AP1903 for 10 days (+) or left unchallenged (−). DNA was digested with XbaI, separated by electrophoresis and probed with an FKBP–Fas-specific probe. Lane 1, untransduced control lymphocytes; lanes 2 and 3, LVVFas-transduced lymphocytes; lanes 4 and 5, donor A LV′VFas-transduced lymphocytes; lanes 6 and 7, donor B LV′VFas-transduced lymphocytes; lane 8, control plasmid. Arrows indicate the size of the expected band (1627 bp) and a band truncated by approximately 300 bp.