Man2, a1 and Ricin Resistance in ES Cells Figure 5. Mannose, galactose and sialic acid specific lectin binding analysis. Histograms of differences in Mean Fluorescence Intensity of lectin binding to either AB2.2, NN5 or ricin resistant F10 cells. A, Mannose specific lectins ConA, PSA and LCA represent Concanavalin A, Pisum sativum agglutinin and Lens culinaris agglutinin respectively. B, Galactose specific lectins PNA, GSL1 and ECL represent Peanut agglutinin, Griffonia (Bandeiraea) simplicifolia lectin 1 and Erythrina cristagalli lectin respectively. C, Sialic acid specific lectins MAL1, EBL (SNA) and MAL11represent Maackia amurensis lectin 1, Sambucus nigra lectin and Maackia amurensis lectin 11 respectively. doi:10.1371/journal.pone.0022993.g005 Materials and Methods Cell culture AB2.2, NN5 and NGG5.3 ES cell lines. These ES cell lines have been described previously [8]. The NN5 and NGG5.3 ES cell lines are Blm-deficient. The AB2.2 and NN5 cells were maintained on gelatin coated plastic in Glasgow’s minimal essential medium (GMEM) supplemented with 10% foetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, non- essential amino acids, 1024 M b-mercaptoethanol and 500–1000 units of leukocyte inhibitory factor (LIF)/ml. NGG5.3 ES cells were maintained as described previously [27]. Briefly, ES cells were maintained on gamma-irradiated SNL76/7 feeder cell layers in Dulbecco’s modified Eagle’s medium supplemented with 15% foetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, 40 mg/ml streptomycin, and 1024 M b-mercaptoethanol. Cells were cultured at 37uC with 5% carbon dioxide. Ricin resistant F10 cells. Clones arising from exposure to ricin were grown on SNL76/7 feeder cells until large enough to pick into 96 well plates also bearing feeder layers. The clones were expanded through 24 and 6 well plates until sufficient numbers were obtained to freeze down and prepare DNA for PCR analysis. Adaptation of F10 to feeder-free conditions. To allow comparative experiments to be performed, feeder dependent F10 cells were trypsinised and washed once in NN5/AB2.2 medium prior to plating into gelatinized 90 mm dishes at a 1 in 8 dilution. Subsequent sub-culturing was performed every two days at the same dilutions until all feeder cells were depleted. Gene-trap vectors construction The PiggyBac (PB) transposon vector containing 313 bp of the 59 inverted terminal DNA repeat (TR) and 235 bp of 39 inverted TR was provided by Dr. Xiaozhong Wang (North Western Univer- sity). The PB59 and PB39 inverted repeats were PCR amplified and cloned upstream and downstream from the adSA- or En2SA- b-geo gene-trap cassette and named PBGTVs. Generation of gene-trap mutations A single-cell clone of NGG5.3 ES cells was isolated and expanded to minimize background mutations. Five million cells were co-electroporated with 1 mg of gene-trap vector and 20 mg of a PBase expression plasmid pCAGGS-PBase, in which the native version of PiggyBac transposase coding sequence is under the control of the CAG promoter. After electroporation the cells were plated onto 90-mm feeder plates and G418 selection (180 mg/L active ingredient) was initiated 24 h later and continued for 8 days until individual ES cell clones were visible. The G418 resistant clones within each plate were pooled and expanded for 4 days before initiating ricin selection. PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e22993