Titrated dhs-specific mRNA resulted in a limit of detection of 20 ng while eIF-5A-specific mRNA could only be detected at a concentration of 200 ng. Optimal primer
binding was determined for eIF-5A-specific primers at a cDNA concentration of 130 ng and for dhs-specific primers at a cDNA concentration of 650 ng (data not shown). In sum, these data demonstrated that Plasmodium-specific eIF-5A and DHS sequences can in principal be silenced by RNAi. Monitoring in vivo silencing of eIF-5A and DHS in erythrocytic stages after infection of NMRI mice with transgenic schizonts from P. berghei With regard to the in vitro results, we investigated the silencing effect of the expressed DHS-specific and eIF-5A specific shRNAs in an in vivo rodent model of P. berghei
ANKA strain . Infection of NMRI mice with P. berghei ANKA wild type strain leads to experimental cerebral malaria within 6 to Go6983 manufacturer 10 days p. i. although the parasitemia is only in the range of 3–5% infected erythrocytes. In case of the infectious but non lethal phenotype P. berghei strain NK56, the infected mice succumb to high parasitemia within 80 days p.i. without cerebral malaria. In a first step DHS-specific shRNA #176 or eIF-5A-specific shRNA #18 expressed from pSilencer 1.0-U6 click here vector was transfected into schizonts, the late developmental stage of the parasite. These transgenic schizonts were applied to NMRI mice for infection. In vivo gene silencing was monitored in the animals’ erythrocytes at day 2 post infection by RT-PCR as before. Infection with schizonts containing the eIF-5A-specific shRNA #18 vector (Figure 3A lane 2) led to a complete disappearance eFT-508 order of the respective transcripts, at least within the detection level of this assay. By contrast, the eIF-5A sequences were clearly detected
in the erythrocytic stage after infection with schizonts, which were transfected with the dhs-specific shRNA #176 vector (Figure 3A, lane 1). Several control reactions were applied. The RT-PCR reactions of a kanamycin control RNA of 1.2 kb (Figure 3A, lane 5) and that of the recombinant eIF-5A plasmid from P. vivax was monitored, resulting in amplification products of approximately 323 bp and 448 bp, respectively (Figure 3A, lanes 5 and 4). Arachidonate 15-lipoxygenase In parallel we confirmed the quality of the total cellular RNA preparation for the presence of the α-tubulin II sequences, which are expressed in the asexual blood stages of Plasmodium (lane 4). Figure 3 A) Monitoring in vivo silencing of parasitic eIF-5A by RT-PCR in RBCs of infected NMRI mice 2 days post infection. NMRI mice were infected with transgenic schizonts harbouring the expressed shRNA P#18. M1) 1 kb ladder (LifeTechnologies, Karlsruhe, Germany); 1) non-transfected 293T cells 2) EIF-5A-siRNA; 3) A positive control for the quality of cellular RNA is the 548 bp amplificate generated with α-tubulin gene-specific primers from P. berghei; 4) A PCR-control reaction with eIF-5A-gene specific primers from P.