KAHRP dsRNA is transcribed in P. falciparum but does not silence endogenous KAHRP. (A) Schematic of the two exon structure of the endogenous KAHRP locus and the construct pHH/KRNAi, created to generate dsRNA for silencing endogenous KAHRP. Large arrows above pHH/KRNAi indicate direction of transcription and position of oligonucleotides used for PCR or RT–PCR are indicated by small arrowheads. The human DHFR (hDHFR) selectable marker cassette is under the transcriptional control of the CAM 5′ and HRP2 3′ untranslated regions. Promoter regions are symbolized by right-angled arrows and the 3′ untranslated regions necessary for gene expression by a circle. (B) Transgenic 3D7/KRNAi parasites transcribe dsRNA. Total RNA was isolated from asynchronized parental (3D7) or transgenic (3D7/KRNAi) parasites and cDNA was then generated using oligo d(T) or a luciferase specific primer (lucR). RT–PCRs were performed with the oligonucleotides either in the presence (+) or absence (–) of reverse transcriptase. As controls, genomic DNA (gDNA) isolated from 3D7 or pHH/KRNAi DNA was also used. (C) Northern blot analysis of RNA isolated from 3D7 parental or 3D7/KRNAi transgenic parasites across the asexual life cycle (Exp1) or during the stage of parasite development when KAHRP expression is at its peak (Exp2 and Exp3) using probes directed against luciferase (LUC), PF14_0344 or KAHRP confirms the KAHRP hairpin structure is transcribed yet is not capable of destabilizing KAHRP mRNA. The percentage of relative KAHRP expression was calculated by determining the level of KAHRP transcripts relative to that of PF14_0344 for both 3D7/KRNAi and 3D7, with the latter being normalized to 100%. ER, early ring-stages; R, rings; LR, late rings; ET, early trophozoites; LT, late trophozoites.

Expression of antisense mRNA fails to knock-down transient luciferase activity. (A) Schematic of two constructs, pHH/lucS and pHH/lucAS, designed to generate sense or antisense luciferase mRNA in P. falciparum, respectively. The large arrow indicates the direction of transcription. (B) Luciferase activity observed in parasite extracts transiently transfected with various plasmid combinations. The level of firefly luciferase was normalized to the level of R. reniformis luciferase to control for variation in transfection efficiency across experiments. The firefly luciferase activity observed in parasites transfected with pHH/lucS and pCC4/lucS were set at 100%, with all other luciferase activities observed in other transfected parasites containing pHH/lucS or pCC4/lucS expressed relative to these, respectively. No antisense/sense mediated knockdown of luciferase activity can be seen, irrespective of vector source for luciferase expression.

CTRP dsRNA does not silence endogenous CTRP during P. berghei ookinete development. (A) Schematic representation of the endogenous CTRP locus and the construct pCTRPdsRNA used to generate dsRNA for silencing endogenous CTRP. Large arrows above pCTRPdsRNA indicate direction of transcription and position of oligonucleotides used for PCR or RT–PCR are indicated by small arrowheads. The T. gondii DHFR (TgDHFR/TS) selectable marker cassette is under the transcriptional control of the P. berghei DHFR 5′ and DHFR 3′ untranslated regions. Promoter regions are symbolized by right-angled arrows and the 3′ untranslated regions necessary for gene expression by a circle. (B) Transgenic P. berghei 299cl2 transcribe dsRNA. Total RNA was isolated from an in vitro ookinete culture of HP parental and transgenic 299cl2 lines 16-h post-induction and cDNA was then generated using oligo d(T). RT–PCRs were performed with the oligonucleotides shown, either in the presence (+) or absence (–) of reverse transcriptase. As a control pCTRP/dsRNA DNA was also used. (C) Comparison of steady state levels of CTRP and WARP RNA during ookinete development (4–20-h post-induction) by Northern blot analysis. Two independent 299cl2 RNA samples are shown. (D) Ratios of CTRP versus WARP RNA levels in parental HP (two samples) and 299cl2 (three samples). None of the differences are significant (unpaired t-test).

Exogenous introduction of siRNAs fails to knock-down endogenous protein levels. Immunoblots for five genes targeted by siRNAs specific for four (MTRAP 3 only and scrambled negative control) distinct regions of genes known to be tractable (KAHRP, EBA175 and PfRh2a) or intractable (MTRAP and GAP50) to conventional knockout. Despite incubation at concentrations up to 100–200 µg/ml no consistent reduction in protein level can be seen for any gene.