vectors. The piggyBac transposon gene-trap vectors provide more comprehensive genome coverage and they have the additional advantage of seamless reversion by PBase compared with retroviral vectors [10]. In the screen described here, ricin resistant clones were directly selected from libraries of insertionally mutated Blm-deficient ES cells by exposing them to the toxin. A ricin-resistant clone with a homozygous mutation in the mannosidase 2, alpha 1 (Man2a1) gene was identified confirming the utility of PB system for recessive genetic screens. Comparative glycomic analysis and glycosylation inhibition studies revealed that Man2a1 deficiency altered the sugar spectrum on the ES cell surface. Immunogold imaging showed a reduction in ricin entry in Man2a1 deficient cells. Thus the terminal b-galactose moieties are potential target receptors for ricin. Results Screening ES cell transposon libraries for ricin resistant mutants Libraries of ES cells with gene trap mutations were screened in an attempt to identify mutant cells with enhanced resistance to ricin. Prior to screening, a selective (lethal) dose of ricin for ES cells was established for the wild type parental cell line (AB2.2), the Blm-deficient feeder-dependent line (NGG5. 3) and a Blm- deficient mutant line adapted for growth in feeder-free conditions (NN5). A clonal survival assay was performed by exposing the ES cells to a range of ricin concentrations (1–30 pM) for 3 days and counting colonies after 10 days. The lethal dose of ricin for all cell lines was determined to be 10 pM after a 72 hour exposure (Fig. 1A). A series of PiggyBac (PB) transposon-based gene-trap vectors (PBGTVs; Fig. 1B) containing an adenovirus splice acceptor (adSA) or a mouse gene En2 splice acceptor (En2SA) and a b-geo gene-trap cassette flanked by the 59 and 39 PB terminal DNA repeats were used as the gene-trap vectors in this study. The PiggyBac transposon offers the advantage of being reversible, precise excision can be achieved by re-expressing the PB transposase [11,12]. Previous studies [13] have reported that the 59 PB terminal repeat has promoter activity. To avoid any possible influence of such an activity, the gene-trap cassette was placed in the opposite orientation relative to the 59 PB terminal repeat. To maximize the coverage of the genome, five vectors (PBGTVa, V0, 1, 2 and VK) were used in which the coding sequence of b-geo is compatible with splicing from exons with different reading frames [10,12,14]. Independent co-transfections of the PB vectors with PBase were used to generate a gene-trap library in several sectors. Each sector of the library contained approximately 2,000 independent gene trap mutations, selected in G418. The clones constituting each sector were pooled and expanded through at least 14 cell doublings to facilitate the segregation of homozygous mutant clones and then they were selected with ricin. Each library sector generated varying numbers of resistant clones most of which were sister clones. From five sectors, twenty one independent insertion sites were analysed further. Initially the clones were screened to determine if they harboured homozygous mutations by cloning of the host-transposon junction fragments by splinkerette PCR. Two clones, named F10-1 and F10-2 which originated from the same pool, were examined further. In contrast to wild type Blm-deficient ES cell, the gene-trap mutant F10 clones -1 and -2 were still viable after exposure to 20 pM ricin, Figure 1C. The clonal relationship between clones F10-1 and -2 was investigated by Southern blot analysis using a b-geo probe and EcoRV to restrict the genomic Man2, a1 and Ricin Resistance in ES Cells DNA to reveal clone specific transposon-host junction fragments, Figure 1D. Both F10-1 and -2 showed a 5.6 kb fragment, suggesting that they are sister clones. This was confirmed by PCR analysis of the host-transposon junction fragments for both 59 and 39 PB ends, Figure 1E. Hence a single clone (referred to as F10) was taken forwards for further analysis. The ricin resistance gene-trap insertion maps to the Mannosidase 2, alpha 1 (Man2a1) gene Sequence analysis of the host-transposon junction from clone F10 revealed that the PB transposon had inserted into intron 21 of the Mannosidase 2, alpha 1 gene on chromosome 17, Figure 2A. This insertion was confirmed to be homozygous by long range PCR (LR-PCR) and triple primer PCR, Figures 2B and C. LR- PCR from the F10 clone generated a 6.6 kb product which included the whole PB transposon and 428 bp of flanking DNA while the PCR product from the NGG5.3 cell line was a 428 bp fragment amplified from the genomic locus, Figure 2B. The triple primer PCR on F10 genomic DNA generated a 335 bp fragment from the 39 PB-host junction or a 376 bp fragment from the 59 PB- host junction but a wild type fragment was not amplified. The same primer set only amplified the endogenous wild type 428 bp fragment in NGG5.3 (Fig. 2C). Reversion analysis To establish causality between the insertion of the transposon and the ricin resistant phenotype, the transposon was designed to be revertible with PBase. The entire gene trap transposon cassette can be removed with PBase which precisely excises the transposon without leaving a footprint, Figure 3A. The F10 cell line was transiently transfected with PBase and plated at clonal density to isolate revertant clones. These clones were isolated and sib-selected to test their sensitivity to ricin. Four out of 96 tested clones regained ricin sensitivity (Fig. 3B). Analysis of the PB-insertion site in the Man2a1 locus revealed that all four resistant clones had lost the PB transposon from one copy of the locus, restoring one allele to its original wild type sequence and function (Fig. 3C). The ricin sensitivity of the heterozygous mutant clones was evaluated by exposure to a range of ricin concentra- tions. This revealed that the heterozygous clones were indistin- guishable from wild type clones, illustrating that there was no dosage affect in the heterozygous state (Fig. 3D). Ricin resistance conferred by a glycosylation inhibitor Man2a1 is a key enzyme in the Golgi involved in N-linked glycosylation of proteins, a process which involves the sequential cleavage of glucose and mannose residues from the post- translationally added precursor structure, prior to the addition of alternative sugars, Figure 4A. Well characterized inhibitors are available for steps in the glycosylation pathway, Figure 4A (numbered 1–8) [15]. In principle, inhibition of this pathway at an appropriate point could mimic aspects of the ricin resistance phenotype of the F10 mutant. We tested several inhibitors, deoxynojirimycin, deoxymannojir- imycin and swainsonine (#3, #6 and #7 respectively, shown in red in Fig. 4A), which are believed to act prior to or on Man2a1 in the pathway. Swainsonine is a reversible active-site inhibitor of Lysosomal a-mannosidase and blocks the processing of high mannose to complex type oligosaccharides. Its use 4 hours prior to and during ricin exposure protected approximately 10–25% of the exposed cells against ricin over and above inhibitor-free cells, whilst the remaining two inhibitors deoxynojirimycin and deoxymannojirimycin were less effective at all concentrations PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e22993