FIG. 2.

Production of Gal-null porcine cells. (A) Targeting of the GGTA1 allele of PED cells using the pGalGTΔS-loxNeo vector. Arrows indicate PCR bands derived from targeted (T) and wild-type (WT) amplicons for the 5′ (primers F238 and R823) and 3′ (F527 and GR2520) genomic-DNA targeting assays. Similar results were obtained with single cell clones of Gal-null ST-IOWA cells. (B) FACS analysis of GGTA1-targeted ST-IOWA cells. Following targeting of the first allele, Gal-null cells were selected using three rounds of baboon NAb (50 μg/ml) and rabbit complement selection. The Gal status was analyzed by FACS analysis with IB4 lectin. Dark profile, unstained cells; light profile, IB4-stained cells. Similar results were obtained with PED cells. (C) Gal-null ST-IOWA cells are resistant to NAb-mediated lysis. ST-IOWA cells were exposed to purified NAb in the presence of supplementary complement, and cell lysis was determined by cell counting. ▪, Gal-positive ST-IOWA cells; □, Gal-null ST-IOWA cells.