In vivo and in vitro knockdown of FREP2 gene expression in the snail Biomphalaria glabrata using RNA interference - PubMed (original) (raw)
In vivo and in vitro knockdown of FREP2 gene expression in the snail Biomphalaria glabrata using RNA interference
Yiguo Jiang et al. Dev Comp Immunol. 2006.
Abstract
RNA interference (RNAi) is reported here for the first time for Biomphalaria glabrata, the snail intermediate host for the human parasite Schistosoma mansoni. The fibrinogen-related protein 2 (FREP2) gene, normally expressed at increased levels following exposure to digenetic trematode parasites, such as S. mansoni or Echinostoma paraensei, was targeted for knockdown. Double-stranded RNA (dsRNA) corresponding to specific regions of the FREP2 gene was introduced into snails by direct injection into the hemolymph, 2 days prior to exposure to trematodes, or added to co-cultures of B. glabrata embryonic (Bge) cells and E. paraensei sporocysts. After introduction of FREP2 dsRNA, expression levels of FREP2 were significantly reduced, to 20-30% of control values. In addition, we were able to disrupt expression of the house-keeping myoglobin gene, further confirming the feasibility of RNAi for B. glabrata. Cross-reactivity in RNAi has not been observed either among four FREP gene subfamilies or between FREP2 and myoglobin. Establishment of RNAi techniques in B. glabrata provides an important tool for clarifying the function of genes believed to play a role in host-parasite interactions, specifically between B. glabrata and its trematode parasites, including S. mansoni.
Figures
Fig. 1
Effect of timing on knockdown of FREP2 expression in BS-90 snails after exposure to S. mansoni. Open bars show FREP2 expression levels at different days post-exposure (DPE). The symbol “#” indicates a significant elevation (P<0:01) of expression of FREP2 in the sham-injected snails at the given time point post-exposure to the parasite, relative to non-exposed, sham-injected snails. Filled bars show the expression of FREP2 after introduction of dsRNA (5 µl at 1µg/µl). In this experiment snails were exposed to S mansoni at 2 days post-injection (DPI). An “*” indicates a significant reduction (P < 0:05) in FREP2 expression in snails injected with dsRNA compared to sham-injected snails at the same time post-exposure to the trematode.
Fig. 2
Effects of different doses of FREP2 dsRNA on knock-down of FREP2 expression in BS-90 snails after exposure to S. mansoni. Snails were injected with different doses of dsRNA, followed by exposure to 30 S. mansoni miracidia at 2 DPI of dsRNA. RNA was extracted from the snails at 4 DPE. Five snails (n = 5) were used in each experiment. The symbols “*” and “**” indicate P < 0:05 and P < 0:01, respectively.
Fig. 3
Northern blot analysis showing RNAi effect in BS-90 snails. Lane 1 shows RNA from control snails at 4 days post sham-injection. Lanes 2 and 3 represent RNA from snails that were either sham-injection (lane 2) or injected with dsRNA (lane 3), followed by exposure to S. mansoni for 2 days (i.e., 4 DPI). For each lane, total RNA was pooled from the whole bodies of 10 snails. The upper image shows the intensity of FREP2 probe binding to mRNA. The lower photos show the staining intensity of rRNA from lanes, as loading controls. Approximate molecular sizes (kilobase) are indicated on the right.
Fig. 4
Effect of timing on knockdown of FREP2 expression level in M line snails after exposure to E. paraensei. Open bars show FREP2 expression levels in M line snails. The symbol “#” indicates a significant elevation (P < 0:01) of expression of FREP2 in the sham-injected snails at the given time point post-exposure to E. paraensei, relative to non-exposed, sham-injected snails. Filled bars show the expression of FREP2 after introduction of FREP2 dsRNA (5 µl at 1µg/µl). Snails were exposed to E. paraensei at 2 DPI. An “*” denotes a significant reduction (P < 0:05) in FREP2 expression in snails injected with dsRNA, compared to sham-injected snails at the same time post-exposure to the trematode.
Fig. 5
Induction of FREP2 expression in Bge cells after exposure to different numbers of E. paraensei miracidia. Bge cells were seeded at 2 × 105 cells per well in 500 µl medium, one day prior to addition of parasites. Parasites were in inserts to maintain separation between them and the Bge cells. RNAs was extracted from the cells 4 day after addition of parasites. An “*” indicates significance when compared to control (P < 0:05). Three wells were used for each treatment.
Fig. 6
Knockdown of FREP2 expression level in Bge cells after exposure to E. paraensei. Bge cells were seeded as described in Fig. 5. After one day of cell culture, 5 µg FREP2 dsRNA was applied to the culture medium. Two thousand miracidia were added 2 days after the dsRNA. Bge cells were harvested at different DPE, i.e., 1, 2, 4 and 6 days for RNA extraction. Open bars show FREP2 expression levels in the Bge cells at different DPE. The symbol “#” indicates significant elevation (P < 0:01) of expression of FREP2 in the sham-injected snails at the given time point post-exposure to the parasite, relative to the non-exposed cells. Filled bars show the expression of FREP2 after introduction of FREP2 dsRNA (5 µl per well). An “*” denotes a significant reduction (P < 0:05) in FREP2 expression in the cells in which the FREP2 dsRNA was added compared to cells at the same time post-exposure to the trematode, but with no involvement of RNAi. The analysis was based on three independent wells for each experiment.
Fig. 7
Expression of myoglobin in BS-90 snails was reduced after introduction of myoglobin dsRNA (5 µl at 1µg/µl). The sham-injection (control) was conducted in each experiment, and the expression value is presented by a solid horizontal line indicated by 1.0 in the figure. An “*” indicates a significant reduction (P < 0:05) in myoglobin expression relative to non-RNAi treated snails at 2, 3 and 4 DPI.
Fig. 8
Specificity of RNAi effects in B. glabrata. (A) RNAi cross-reactivity did not occur between myoglobin and FREP2 in BS-90 snails. The injection of FREP2 dsRNA resulted in a significant reduction in FREP2 expression (*P < 0:01) compared to sham-injected controls, whereas _myoglobin_ expression was not significantly altered. Conversely, the injection of _myoglobin_ dsRNA resulted in a significant reduction in _myoglobin_ expression (*_P_ < 0:01) compared to sham-injected controls, whereas _FREP2_ expression was not significantly reduced. As before, all snails were exposed to _S. mansoni_ at 2 DPI of dsRNA or of nuclear-free water (sham-injection). qPCR data were collected at 4 DPE (i.e., 6 DPI). (B) Expression level of _FREP_ genes (_FREPs 2, 3, 4, 6, 7_ and _13_) in BS-90 snails after injection of dsRNA of _FREP2_ (5 µg per snail). There were two groups of snails, one injected with _FREP2_ dsRNA (5 µg) and the other sham-injected as a control. Five snails were analyzed in each group by qPCR. We divided the expression values for each _FREP_ gene by their corresponding control expression value. Accordingly, the control expression for each gene is 1 (presented by a solid horizontal line indicated by 1.0 in the figure). Values <1 represent downregulation and values > 1 represent upregulation. *P < 0:01.
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