Haploid genetic screening to reveal factors required for GPI biosynthesis using aerolysin sensitivity.

<p>A. Structure of the gene trap vector and strategy for enrichment of GPI-negative mutant cells. Retrovirus-based gene trap vectors containing splice acceptor site (SA) and polyadenylation signal (PolyA) were infected into HAP1 cells. The 3′ flippase recognition target (FRT) site of the provirus is copied into the 5′ long terminal repeat (LTR). Mutagenized cells (6 × 10⁷ cells) were selected with 6 μg/ml blasticidin for 1 week. To eliminate the possibility that GPI-negative mutant cells were lost during the screening procedure, the 4 times of infected cells (2.4 × 10⁸ cells) were used for the treatment with 0.2 nM proaerolysin for 1 day. After the first treatment, the survived cells were harvested and cultured in new plates. Then, 6 × 10⁷ cells were treated with 0.2 nM proaerolysin for 1 day again. Resistant cells were proliferated and stained with an anti-CD59 antibody and CD59-negative cells were further enriched by cell sorting. Ψ, packaging signal; PGKpro, PGK promoter; CMV pro, CMV promoter; BSD, blasticidin S-deaminase gene. B. Sensitivity of HAP1 cells to proaerolysin. HAP1 cells were treated with indicated concentrations of proaerolysin (nM) for 3 h. After changing to medium without aerolysin, cell viability was measured by the WST-1 assay and shown in % viability. Viability of cells without proaerolysin treatment was 100%. Data are means ± standard deviation (n = 4).