Man2, a1 and Ricin Resistance in ES Cells Figure 3. PBase reversion of mannosidase 2, alpha 1 mutation. A, illustration of the allele bearing the Man2a1PiggyBac insertion before and after reversion with PBase. B, methylene blue staining of four revertant heterozygous clones, and the original ricin resistant F10 clone, either before or after G418 or 20 pM ricin exposure. C, triple primer PCR demonstrating heterozygosity after reversion. D, viability as percentage of control calculated from neutral red staining of AB2.2, NN5, F10 Man2a1 homozygous mutant and revertant cells after exposure to different concentrations of ricin (0 or 1–30 pM). doi:10.1371/journal.pone.0022993.g003 b-linked galatose residues which are recognised by ricin. The libraries used for this screen were generated in Blm-deficient ES cells which facilitates the segregation of homozygous mutants from pools of single allele mutations. This genetic background has supported several successful screens [8,9,18]. The mutation libraries used for this screen were generated using the PiggyBac transposon to insert a conventional gene-trap cassette into the ES cell genome [10]. One advantage of PiggyBac over other insertional mutagens is the fully revertible nature of the mutations, since re- expression of PBase seamlessly removes the PiggyBac transposon from the genome. The ricin-resistant mutant identified here could be reverted to a sensitive state by excision of the PB gene-trap transposon from one of the two copies of the Man2a1 gene. Our screen for ricin-resistant mutants from a PiggyBac gene-trap ES cell library has provided direct access to a gene required for ricin intoxication. To further our understanding of the mechanism of resistance we investigated the cell surface lectin binding properties and also conducted glycomic analyses on the mutant clones. As a confirmation of the direct involvement of Man2a1 in ricin resistance, we treated na¨ıve ES cells with inhibitors of N- linked glycosylation and found that Swainsonine conferred significant resistance to ricin exposure. Meager and colleagues [19], have previously described ricin resistant (RicR) clones of baby hamster kidney cells. A selection of these proved to be resistant due to an inability to bind ricin. Metabolic labelling revealed that these clones express only hybrid and high mannose-type N-linked oligosaccharides rather than the complex sugars found in the parental BHK cells [20,21]. Use of the mannose specific lectin, Concanavalin A, indicated that mannose residues were more abundant on those cells. Some of the RicR cells were deduced to be deficient in Man2a1 activity but the causal defect was never genetically confirmed [22]. These data closely matched our observations on the ricin-resistant ES cell clone identified in our study which exhibited greatly increased binding of PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e22993