Polyethyleneimine (PEI) mediated siRNA gene silencing in the Schistosoma mansoni snail host, Biomphalaria glabrata.

An in vivo, non-invasive technique for gene silencing by RNA interference (RNAi) in the snail, Biomphalaria glabrata, has been developed using cationic polymer polyethyleneimine (PEI) mediated delivery of long double-stranded (ds) and small interfering (si) RNA. Cellular delivery was evaluated and optimized by using a 'mock' fluorescent siRNA. Subsequently, we used the method to suppress expression of Cathepsin B (CathB) with either the corresponding siRNA or dsRNA of this transcript. In addition, the knockdown of peroxiredoxin (Prx) at both RNA and protein levels was achieved with the PEI-mediated soaking method. B. glabrata is an important snail host for the transmission of the parasitic digenean platyhelminth, Schistosoma mansoni that causes schistosomiasis in the neotropics. Progress is being made to realize the genome sequence of the snail and to uncover gene expression profiles and cellular pathways that enable the snail to either prevent or sustain an infection. Using PEI complexes, a convenient soaking method has been developed, enabling functional gene knockdown studies with either dsRNA or siRNA. The protocol developed offers a first whole organism method for host-parasite gene function studies needed to identify key mechanisms required for parasite development in the snail host, which ultimately are needed as points for disrupting this parasite mediated disease

New Cationic Lipids Form Channel-Like Pores in Phospholipid Bilayers

Two representatives of a new class of cationic lipids were found to have high pore-forming activity in planar bilayer membranes. These molecules, called BHHD-TADC and BHTD-TADC, have qualitatively similar effects on phospholipid membranes. Addition of 2.5–5 μM of either of them to the membrane bathing solutions resulted in formation of long-lived anion-selective pores with conductance in the range 0.1–2 nS in 0.1 M KCl. Pore formation was found to be dependent on the potential applied to the membrane. When negative potential was applied to membrane at the side of addition, the rate of pore formation was much lower compared to when the positive potential was applied. Dependence of pore formation on compound concentration was highly nonlinear, indicating that this process requires assembly of molecules in the membrane. Addition of any of these compounds on both sides of the membrane increased the efficiency of pore formation by one to two orders of magnitude. Pore formation was strongly pH dependent. Although pores were formed with high efficiency at pH 6.5, only occasional fluctuations of membrane conductance were observed at pH 7.5. Possible mechanisms of new compounds biological activity are discussed

Snail organs transfected with or without the fluorecent siRNA visualized by either light or fluorescence microscopy.

<p><i>Panels A and C</i>: Images of snail hepatopancreas and ovotestis tissues of juvenile snails that were either soaked in fluorescent siRNA (A) for 72 hrs or left untreated (C) viewed without fluorescence (10× magnification). <i>Panels B and D</i>: The same images of hepatopancreas and ovotestis regions shown in panels A and C, subjected to fluorescence microscopy as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001212#s2" target="_blank">Materials and Methods</a>. Note the very weak flourescence (red stain) in the hepatopancreas (B) indicating less uptake of the fluorescent siRNA into this tissue occurs without PEI (10× magnification).</p

Time course for optimum gene knockdown after soaking snails in either dsRNA/PEI or siRNA/PEI nanoparticles.

<p>(A) Relative expression of Cathepsin B gene in juvenile snails soaked for between 1 and 4 days in CathB dsRNA/PEI. (B) Relative expression of peroxiredoxin gene in snails soaked for between 1 and 4 days in Prx dsRNA/PEI. (C) Relative expression of Cathepsin B gene in snails soaked for 1 to 4 days in CathB1-siRNA/PEI. Note in all time course studies that the optimum knockdown of transcripts occurs at day 3 of incubating in either dsRNA/PEI or siRNA/PEI complexes.</p

Analysis of Cath B gene expression by real time qPCR.

<p>(A) Relative expression of Cathepsin B gene in CathB1 siRNA/jPEI, CathB2siRNA/jet PEI, ‘mock’ CathB siRNA, CathB1siRNA, and PEI treated snails <i>versus</i> untreated snails. Treatments were done at room temperature for 72 hrs. (B) Relative expression of Cathepsin B gene in CathB dsRNA/PEI, Myo dsRNA/PEI, CathB dsRNA, and PEI treated <i>versus</i> untreated juvenile snails. Treatments were done at room temperature for 72 hrs.</p

Analysis of Prx expression by real time qPCR and ELISA.

<p>(A) Relative expression of peroxiredoxin gene in Prx dsRNA/PEI, Myo dsRNA/PEI, Prx dsRNA alone and PEI treated snails <i>versus</i> untreated snails. Treatments were done at room temperature for 72 hrs. (B) Prx protein suppression analyzed by ELISA as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001212#s2" target="_blank">Materials and Methods</a> with recombinant <i>B. glabrata</i> Prx antibody. Each bar represents the geometric mean end point titre ± standard error (SE) of each experimental group as indicated on the y-axis.</p

Snail organs transfected with the jPEI/fluorescent siRNA nanoparticles visualized by light and fluorescence microscopy.

<p><i>Panels A and C</i>: Images of hepatopancreas and ovotestis regions of juvenile snails that were soaked either in jPEI/Alexa 555 siRNA for either 24 or 72 hrs viewed without fluorescence (10× magnification). <i>Panels B and D</i>: Images of the hepatopancreas and ovotestis tissues shown in panels A and C, subjected to fluorescence microscopy as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001212#s2" target="_blank">Materials and Methods</a>. Note the intense flourescence (red stain) in the hepatopancreas compared to the ovotestis, indicating preferential uptake of the jPEI/fluorecent siRNA nanoparticles into this tissue.</p