Genetic Mapping Reveals that Sinefungin Resistance in Toxoplasma gondii Is Controlled by a Putative Amino Acid Transporter Locus That Can Be Used as a Negative Selectable Marker

ABSTRACTQuantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasiteToxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDRrand type 10 VAND-SNFr. The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of theSNR1gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies inT. gondii.

324 NEGATIVE SELECTION DOES NOT FURTHER INCREASE THE EFFICIENCY OF A PROMOTER TRAP

Every year more than 100 000 people need organ transplants and in 2009 there were only 20 139 deceased organ donors. Genetically modified swine may be able to fill this unmet clinical need. It is clear that multiple transgenes will be required to make porcine organs or cells compatible with human recipients. Efficiencies in production of xenotransplantation pigs could be gained if multiple transgenes could be sequentially stacked at a single locus. The larger purpose of this project is to evaluate a site-specific recombination system as a tool to sequentially add multiple transgenes to the porcine α galactosyltransferase (α1,3-Gal) locus. One xenotransplantion related transgene, human decay accelerating factor (hDAF), is likely to be a part of any successful project because hDAF can prevent acute rejection by deactivating the complement system. To generate transgenic pigs that express an hDAF transgene that is genetically linked to an α1,3-Gal disruption, an α1,3-gal gene targeting vector was constructed with homologous arms that were 4.8 kb (5′ arm) and 1.8 kb (3′ arm) and were derived from genomic sequence from intron 8 and exon 9 of the α-Gal locus. Within exon 9, an IRES-Neo cassette and a CAG-hDAF cassette were inserted. A Phi-C31 AttB site was also included in the construct to later receive additional transgenes via a site-specific recombination system. This vector (pBB7) is designed to utilise a promoter-trap strategy as a method of enrichment for targeting events. We hypothesised that further enrichment could be obtained by the addition of a negative selectable marker. The goal of the experiment presented here was to evaluate this hypothesis by adding a truncated diphtheria toxin cassette (TdT) to pBB7 as a negative selection directed against random integration events. Two additional plasmids were constructed that harbored TdT, pBB8.1 (single TdT addition) and pBB8.2 (addition of 2 TdT cassettes). Four treatments (Trt) were performed in triplicate: Trt1, pBB7; Trt2, pBB8.1; Trt3, pBB8.2; and Trt4, pBB7 co-transfected with TdT. Targeting efficiencies were 13.9, 11.1, 5.6, and 6.1%, respectively. Successful targeting was assessed by PCR with primers specific for a targeting event. The first pair of primers flanked the 5′ arm of the construct and the neomycin resistance gene cassette. The second pair of primers flanked the 3′ arm of the construct and hDAF gene cassette. Although all treatments produced targeting events at very high rates, TdT inclusion in the vector (1 or 2 copies) or as a co-transfectant did not further improve the promoter trap strategy. It is concluded that a negative selectable marker may not further improve the efficiencies provided by a promoter trap strategy.