The PfFCKpnIR control primer binding site is indicated by an arrow labeled as “c.” The locations of SpeI and HindIII restriction sites and the Southern blot probe hybridizing regions are indicated on the genomic DNA line. The locations of BglIIPfFCF and 3UTRpbDT_glmSR primer binding sites for integration-specific PCR assay are indicated by arrows labeled “a” and “b,” respectively. (B) Schematic diagram of the PfFC gene region in transgenic parasites with integrated PfFC_ glmS plasmid (all elements drawn to scale). The locations of SpeI and HindIII restriction sites and the Southern blot probe hybridizing region are indicated on the genomic DNA line. Plasmid backbone (not to scale) is depicted as a dashed blue line. berghei dihydrofolate reductase-thymidylate synthase gene terminator ( PbDT). Functional elements in transfection plasmids are depicted as boxes: the blasticidin-S-deaminase (BSD) selection marker gene, camodulin gene promoter (CAM), histidine-rich protein two gene terminator (hrp2), PfFC homologous targeting sequence ( PfFC), GFP gene (GFP), glmS riboswitch ( glmS), and P. The PfFC_ glmS transfection plasmid integrated into the PfFC locus in transgenic parasites via single-crossover recombination, indicated by the dashed lines. The PfFC annotated gene model exons are indicated by red boxes. (A) Schematic diagram of the Plasmodium falciparum 3D7 genomic region encompassing the PF3D7_1364900 ( PfFC) gene, drawn to scale. Supplemental Information 2: Transgenic modification of the PfFC gene. The migrations of one kb+ DNA ladder (Invitrogen) bands are indicated on the left. falciparum 3D7 parental parasite lane 2, PfDHS_ glmS transgenic parasite lane 3, PfDHS_M9 transgenic parasite. PCRs were conducted with genomic DNA templates from different parasites: lane 1, P. (E) Agarose gel of PCR products for integration-specific PCR assay.
falciparum 3D7 parental parasite genomic DNA lane 2, PfDHS_ glmS transgenic parasite genomic DNA lane 4, PfDHS_M9 transgenic parasite genomic DNA. DNA samples: lane 1, PfDHS_ glmS plasmid DNA (7155 bp) P. The membrane was hybridized with a DIG-labeled DNA probe synthesized by PCR using primers pGFP_glmS_DHSF and GFP_glmS_DHSR. DNA samples were digested with BamHI and AflII, separated by agarose electrophoresis and blotted onto a Hybond N+ nylon membrane (GE healthcare). The locations of DHS_intF and 3UTRpbDT_glmSR primer binding sites for integration-specific PCR assay are indicated by arrows. The locations of BamHI and AflII restriction sites and the Southern blot probe hybridizing regions are indicated on the genomic DNA line. (C) Schematic diagram of the PfDHS gene region in transgenic parasites with integrated PfDHS_M9 plasmid (all elements drawn to scale).
(B) Schematic diagram of the PfDHS gene region in transgenic parasites with integrated PfDHS_ glmS plasmid (all elements drawn to scale).
Functional elements in transfection plasmids are depicted as boxes: the blasticidin-S-deaminase (BSD) selection marker gene, camodulin gene promoter (CAM), histidine-rich protein two gene terminator (hrp2), PfDHS homologous targeting sequence ( PfDHS), GFP gene (GFP), glmS riboswitch (wild-type or M9 variant glmS), and P. The transfection plasmids PfDHS_ glmS and PfDHS_M9 integrated into the PfDHS locus in transgenic parasites via single-crossover recombination, as indicated by the dashed lines. The locations of AflII restriction sites and the Southern blot probe hybridizing region are indicated on the genomic DNA line. The PfDHS annotated gene model exon is indicated by a red box. (A) Schematic diagram of the Plasmodium falciparum 3D7 genomic region encompassing the PF3D7_1412600 ( PfDHS) gene, drawn to scale. Supplemental Information 1: Transgenic modification of the PfDHS gene.
0 kommentar(er)
